WO2000050902A2 - High throughput assay based on the use of a polypeptide binding pair - Google Patents
High throughput assay based on the use of a polypeptide binding pair Download PDFInfo
- Publication number
- WO2000050902A2 WO2000050902A2 PCT/GB2000/000669 GB0000669W WO0050902A2 WO 2000050902 A2 WO2000050902 A2 WO 2000050902A2 GB 0000669 W GB0000669 W GB 0000669W WO 0050902 A2 WO0050902 A2 WO 0050902A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- polypeptides
- immobilised
- peptide
- modification
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
Definitions
- the present invention relates to a method for detecting or monitoring biologically significant molecules.
- the invention relates to the use of immobilised polypeptides which are capable of giving rise to a signal, this signal changing in relation to the modification state of the polypeptides, and thereby allowing the modification state of the polypeptides to be assayed by monitoring of said signal.
- Post-translational modification of proteins has long been regarded as central to the regulation of cellular processes. Modification of proteins may affect their biological activities, or may affect their binding affinities for other cellular components, or may mark a protein for degradation, or have many other effect(s) on the protein. However, monitoring the post-translational modification state of proteins has been fraught with difficulties.
- monitoring the post-translational modification of various proteins has been accomplished in the past by extension of those techniques outlined above.
- monitoring of ubiquitination of proteins has been carried out by extracting proteins from the sample to be tested, immunoprecipitating the protein of interest, Western-blotting the immunoprecipitated protein, and then developing the Western-blot using an anti-ubiquitin antibody.
- this is a complicated procedure.
- there are many different isoforms of ubiquitin and so repetition of the assay with antibodies against all the various forms of ubiquitin can increase the effort involved many-fold.
- the present invention seeks to overcome such difficulties.
- a method for analysing a sample comprising the steps of: -
- association of the polypeptides is detectable, and (ii) modification of at least one of the polypeptides results in modulation of the association;
- sample refers to a collection of inorganic, organic or biochemical molecules which is either found in nature (e.g., in a biological or other specimen) or in an artificially-constructed grouping, such as agents which might be found and/or mixed in a laboratory.
- a sample may be either heterogeneous or homogeneous.
- such a sample may contain one or more agents which are capable of modifying a polypeptide.
- An immobilised polypeptide is one which is bound to a solid phase support.
- This binding may be covalent or via ionic bonding, hydrogen bonding, van-der-waals forces or any other non-covalent attachment, including antibody-antigen attachment, Ni-NTA attachment, avidin-biotin pairing and the use of GST tags.
- the solid phase may be a membrane, for example supported nitrocellulose, a bead, for example an agarose, glass or Sepharose bead, a magnetic bead, a plastic substrate such as an ELISA dish or other plate, or may be a BIAcore chip or other silicon based chip.
- the polypeptide is bound to the support in such a way that it is at least partly free in solution.
- the polypeptide may be bound to the support via an N- or C- terminal linkage, for example via a C-terminal cysteine residue.
- Modification of a polypeptide may include proteolysis (proteolytic cleavage), phosphorylation, dephosphorylation (phosphatase), acylation (for example fatty acylation such as famesylation, geranylgeranylation, myristoylation, palmitoylation), glycosylation, ubiquitination, prenylation, sentrinisation, ADP-ribosylation, or the reversal of these processes where these are possible.
- the immobilised polypeptide may be a substrate for one or more of these enzymatic activities.
- the term 'modification' as used herein may also include the binding of one or more molecules of the test sample to a polypeptide.
- one of the polypetides is bound to a solid support.
- This polypeptide is referred to as the "immobilised polypeptide”.
- Polypeptides capable of binding to, or bound to, the immobilised polypeptide are referred to as "binding partner" polypeptides.
- the association between the immobilised polypeptide and the binding partner polypeptide may be measured by any suitable means.
- a signal may be generated which may be dependent on the interaction of two polypeptides, one or both of which may be labelled. If the polypeptides interact, excitation of a fluorophore on one may result in Fluorescence Resonance Energy Transfer (FRET), and emission of light of a particular measurable wavelength from a fluorophore on the other.
- FRET Fluorescence Resonance Energy Transfer
- the assay may be configured in such a way that modification of the immobilised polypeptide affects its capacity to bind the binding partner polypeptide. Therefore, by measuring the binding of the partner polypeptide, the modification state of the immobilised polypeptide may be inferred.
- the assay is configured in such a way that biologically significant events are able to influence the signal.
- the first or second polypeptides is exposed to a sample containing one or more agent(s) which modifies the polypeptide(s), then this modification may be detected via its effect on the binding or association of the polypeptides.
- the invention therefore provides a method according to the first aspect described above, further comprising the step of exposing at least one of the polypeptides to an agent capable of modifying the polypeptide(s).
- an agent capable of modifying the polypeptide(s).
- the term 'agent' is understood to mean a substance or group of substances which are capable, either singly or in combination, of bringing about modification of a polypeptide.
- Such an agent may be an enzymatic activity, or may be a chemical entity, or may be a mixture of one or more such entities.
- the present invention provides a method for detecting and/or measuring post-translational modification of a substrate polypeptide by a sample.
- the invention provides a polypeptide pair comprising a first polypeptide immobilised to a support, and a second polypeptide bound to the first polypeptide, wherein:
- the invention provides a method for detecting or monitoring the activity of a modulator of a polypeptide modifying agent, comprising the steps of:
- At least one of the polypeptides is susceptible to modification, and (ii) the first and second polypeptides are capable of binding to each other, and modification of one or both of the polypeptides by the modifying agent results in modulation of the binding of the polypeptides to each other;
- the reference binding modulation is that degree of modulation, whether an increase or decrease in binding affinity or avidity, which is observed in the presence of a peptide modifying agent.
- the reference modulation need not be determined de novo for every analysis. It is sufficient for a reference modulation to be established such that the activity of potential modulators of peptide modifying agents can be assessed.
- Modulators of peptide modifying agents may be any agents capable of upregulating or downregulating agents which themselves modify polypeptides.
- a modulator may be an agent capable of potentiating or reducing the activity of a kinase or a phosphatase, a ubiquitin conjugating enzyme, a fatty acylation enzyme or other enzyme capable of modifying polypeptides.
- the invention further provides support comprising one or more immobilised polypeptides, wherein at least one of the immobilised polypeptides is susceptible to modification, said modification being detectable by a method comprising:
- Figure 1 is a graph showing detection of immobilised phosphorylated TCR ⁇ peptide (peptide 1) using ZAP-GFP.
- Figure 2 is a bar chart showing phosphorylation of TCR ⁇ peptide 2 by Src kinase detected by ZAP-GFP binding.
- Figure 3 is a graph showing the phosphorylation of TCR ⁇ peptide by different amounts of Src enzyme deteced by the fluorescence of bound ZAP-GFP.
- Figure 4 is a time course showing Src kinase phosphorylation of TCR ⁇ peptide, detected by the fluorescence of bound ZAP-GFP.
- Figure 5 illustrates the measurement of Src inhibition by staurosporine.
- IC o 64 nM.
- Figure 6 is a time course showing YOP dephosphorylation of phosphorylated TCR ⁇ peptide (peptide 1), detected by the decrease in fluorescence of bound ZAP-GFP.
- Figure 7 shows the dephosphorylation of TCR ⁇ peptide (peptide 1) by different amounts of YOP, measured by the decrease in fluorescence of bound ZAP-GFP.
- Figure 9 shows the phosphorylation of TCR ⁇ peptide 2 by a crude Fyn kinase preparation detected by the fluorescence of bound ZAP-GFP
- Figure 10 shows a Src phosphorylation of SHPS-1 peptide, detected with (SHP2-GFP).
- Figure 11 shows a concentration curve for Src phosphorylation of SHPS-1 peptide (detected with SHP2-GFP).
- Figure 12 shows a time course for Src phosphorylation of SHPS-1 peptide (detected with SHP2-GFP).
- Figure 13 shows a staurosporine inhibition curve for Src phosphorylation of SHPS-1 peptide (detected with SHP2-GFP).
- Figure 14 shows the detection of Src phosphorylation of peptide 2 using ZAP-FJ labelled with fluorescein.
- Figure 15 shows the immobilisation of increasing concentrations of TCR-4HA to 5HB-biotin on a Neutravidin plate. Phosphorylation by Src. Detection by Zap70GFP or Antibody.
- Figure 16 shows the inhibition of Src kinase by Staurosporine detected using an immobilised assay with TCR ⁇ chain substrate and ZAP-GFP binding partner
- Figure 17 is a flowchart illustrating a possible configuration of an assay of the invention.
- Figure 18 is a diagram illustrating two possible configurations of an assay of the invention.
- Figure 19 is a diagram illustrating a possible configuration of an assay of the invention.
- Figure 20 is a diagram showing the proposed structure of a coiled coil reporter for use in an assay of the invention.
- binding of a first polypeptide to a second polypeptide is dependent upon modification, which modification may occur on one or more polypeptides.
- a polypeptide is susceptible to "modification" if it is capable of serving as a substrate for one or more modifying enzymes in accordance with the present invention.
- the polypeptide may be susceptible to digestion by a specific protease enzyme, and preferably only susceptible to digestion by a specific protease enzyme. This facilitates the reduction of non-specific or background proteolysis and the use of the invention to assay specific proteolytic events.
- a polypeptide may be susceptible to phosphorylation by a specific kinase enzyme, and preferably only be susceptible to phosphorylation by a specific kinase enzyme. This facilitates the use of the invention to assay specific kinase activities.
- a polypeptide may also be susceptible to "modification" by entities other than enzymes. One or more chemical agent(s) may modify the polypeptide, either singly or in combination.
- modification preferably occurs at a suitable site, which may be engineered into the one or more of the polypeptide(s) - an "engineered site” - or may be naturally present in one or more of the polypeptide(s) - a "natural site".
- a suitable site which may be engineered into the one or more of the polypeptide(s) - an "engineered site” - or may be naturally present in one or more of the polypeptide(s) - a "natural site”.
- modification site refers to an amino acid sequence which is recognised by (i.e., is a recognition site for) a modification enzyme. It is contemplated that a site comprises a small number of amino acids, typically from 2 to 10. less often up to 30 amino acids, and further that a site comprises fewer than the total number of amino acids present in the polypeptide.
- An engineered modification site may be placed within a polypeptide of the invention at a position such that formation of an association between the isolated polypeptide and its binding partner is dependent upon the intactness of the site, Preferably, it does not overlap with an amino acid which is part of a label, to ensure that any detectable interaction is due to dissociation and not label loss.
- the position at which an engineered site is to reside may initially be determined by random placement of the site within the polypeptide, followed by testing by methods described herein of the ability of the polypeptide to associate with its intended binding partner(s), or not, depending upon the intactness or otherwise of the site.
- a pair of polypeptides, of which at least one comprises a site so placed that association of the polypeptides is dependent on cleavage at this site, is useful in the present invention.
- a polypeptide which is susceptible to "modification” may be subject to or may be the target of modification by any “modification enzymes” such as proteases, kinases, phosphatases, farnesyl transferases, ADP-ribosylating enzymes, glycosylating enzymes, geranylgeranyl transferases, ubiquitinating enzymes, sentrinisation enzymes, fatty acylation enzymes, such as prenylating enzymes, myristoylation enzymes and palmitoylation enzymes, or any other polypeptide modifying enzyme.
- modify enzymes such as proteases, kinases, phosphatases, farnesyl transferases, ADP-ribosylating enzymes, glycosylating enzymes, geranylgeranyl transferases, ubiquitinating enzymes, sentrinisation enzymes, fatty acylation enzymes, such as prenylating enzymes, myristoylation enzymes and palmitoylation enzymes, or
- Recognition or substrate sites for one or more of these enzymes may be engineered sites or natural sites as described above.
- polypeptide refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulphide bonds.
- Polypeptide refers to an amino acid chain or a fragment thereof, such as a selected region of protein that is of interest in a binding interaction, or a synthetic amino acid chain, or a combination thereof.
- Polypeptide thus refers to an amino acid sequence between about 2 and about 500 amino acids in length, preferably about 4 to about 300, more preferably about 6 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids in length, most preferably about 20 to about 30 amino acids in length.
- amino acids other than naturally-occurring amino acids for example ⁇ - alanine, phenyl glycine or homoarginine, may be included. Commonly-encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L- optical isomer. The D-isomers are preferred for use in a specific context, further described below.
- other peptidomimetics are also useful, e.g. in linker sequences of polypeptides of -l ithe present invention (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids. Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267).
- Naturally-occurring as used herein, as applied to a polypeptide or polynucleotide. refers to the fact that the polypeptide or polynucleotide can be found in nature.
- One such example is a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated form a source in nature. Once the polypeptide is engineered as described herein it is no longer naturally occurring but is derived from a naturally occurring polypeptide.
- Polynucleotide refers to a polymeric form of nucleotides of 2 up to 1,000 bases in length, or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
- the term includes single and double stranded forms of DNA.
- the term is synonymous with "oligonucleotide”.
- the term “associates” or “binds” refers to polypeptides as described herein having a binding constant sufficiently strong to allow detection of binding by a detection means, such as FRET or surface plasmon resonance.
- the polypeptides, when associated or bound are in physical contact with each other and have a dissociation constant (Kd) of about lO ⁇ M or lower.
- Kd dissociation constant
- the contact region may include all or parts of the two molecules.
- the terms “substantially dissociated” or “dissociated” or “substantially unbound” or “unbound” refer to the absence or loss of contact between such regions, such that the binding constant is reduced by an amount which produces a discernible change in a signal compared to the bound state, including a total absence or loss of contact, such that the proteins are completely separated, as well as a partial absence or loss of contact, so that the body of the proteins are no longer in close proximity to each other but may still be tethered together or otherwise loosely attached, and thus have a dissociation constant greater than lO ⁇ M (Kd). In many cases, the Kd will be in the mM range.
- complex refers to the polypeptides, peptides, proteins, domains or subunits in the associated or bound state. More than one molecule of each of the two or more polypeptides may be present in a complex, and molecules other than the two or more polypeptides may also be present in a complex, according to the methods of the invention. In all of these situations, the invention requires merely that the complex should be capable of moving between an associated and a dissociated state in response to modification of a component thereof.
- dissociation refers to the ability of a polypeptide modifying agent, as defined above, to prevent or reverse the association of at least two polypeptides, as defined above, by at least 10%, preferably by 25-50%, highly preferably by 75-90% and, most preferably, by 95-100%) relative to the association observed in the absence of modification under the same experimental conditions.
- the association between the polypeptides may be measured for example by Fluorescent Resonance Energy Transfer (FRET), wherein the immobilised polypeptide harbours a fluorescent label moiety, and the binding partner polypeptide harbours a second such moiety, and where excitation at an appropriate wavelength may result in absorption of photons by one label, followed by FRET, and emission at a second wavelength characteristic of the second fluorophore, this emission being measured and corresponding to the amount of binding partner polypeptide which is associated with the immobilised polypeptide.
- FRET Fluorescent Resonance Energy Transfer
- this association may be measured in one of many other ways which are described more fully below.
- the "detectable signal” or “measuring of association” referred to herein may be any detectable manifestation attributable to the presence of a label and will depend on the means selected for label detection.
- a label will be present on at least two polypeptide components of the bound complex such that association and dissociation thereof can be monitored by measurement of energy transfer between the labels.
- FCS fluorescence correlation spectroscopy
- the label is detected for example by fluorescence correlation spectroscopy (FCS), which relies on the measurement of the rate of diffusion of a label, only a single labelled polypeptide is required.
- FCS detection the labelled polypeptide is the binding partner polypeptide and is detectable in the free, unbound state as being mobile; when complexed to the immobilised polypeptide. it is immobile.
- the “label” preferably comprises a light emitting detection means, and the light emitting detection means advantageously emits light of at least a fluorescent wavelength emission. It is preferred that the light emitting detection means comprises two different fluorophores or fluorescent tags or groups.
- fluorescent tag or “fluorescent group” refers to either a fluorophore or a fluorescent protein or fluorescent fragment thereof, or refers to a fluorescent amino acid such as tryptophan which may be incorporated into a polypeptide.
- fluorescent protein refers to any protein which fluoresces when excited with appropriate electromagnetic radiation. This includes proteins whose amino acid sequences are either natural or engineered.
- the fluorophores comprise fluorescein and tetramethylrhodamine or another suitable pair.
- the label comprises two different fluorescent proteins. It is preferred that fluorescent proteins comprise any protein selected from the group consisting of green fluorescent protein (GFP). blue fluorescent protein, red fluorescent protein and other engineered forms of GFP.
- GFP green fluorescent protein
- the polypeptide comprises a cysteine amino acid through which the label is attached via a covalent bond.
- the label may be attached via a primary amine group such as via a lysine residue.
- the polypeptide is immobilised via cysteine residues, the label is advantageously attached via lysine residues.
- the measuring is performed by fluorescent resonance energy transfer (FRET), fluorescence anisotropy or fluorescence correlation spectroscopy, or by measuring the binding of a fluorescent partner polypeptide to an immobilised polypeptide.
- FRET fluorescent resonance energy transfer
- the fluorescence emitting means comprise two different fluorophores, and particularly preferred that the fluorophores comprise fluorescein and tetramethylrhodamine or another suitable pair.
- the term "appropriate combination” refers to a choice of reporter labels such that the emission wavelength spectrum of one (the “donor” moiety) is within the excitation wavelength spectrum of the other (the “acceptor” moiety).
- the polypeptide multimer is an enzyme which is capable of participating in an enzyme-substrate reaction which has a detectable endpoint.
- the enzyme may be cleaved into two or more components, such that upon multimer formation the components reassemble to form a functional enzyme. Enzyme function may be assessed by a number of methods, including scintillation counting and photospectroscopy.
- the invention may be configured such that the label is a redox enzyme, for example glucose oxidase, and the signal generated by the label is an electrical signal.
- the invention includes detection via the reconstitution of a two subunit enzyme complex or a two subunit photoprotein, such as bacterial luciferase.
- Modification of one or more polypeptides according to the invention is required to inhibit multimer formation and enzyme component assembly, thus reducing enzyme activity.
- an enzyme is used together with a modulator of enzyme activity, such as an inhibitor or a cofactor. Binding of the enzyme and its inhibitor or cofactor results in modulation of enzymatic activity, which is detectable by conventional means.
- a modulator of enzyme activity such as an inhibitor or a cofactor. Binding of the enzyme and its inhibitor or cofactor results in modulation of enzymatic activity, which is detectable by conventional means.
- the invention also encompasses a pair of polypeptides, at least one of which is immobilised, which associate to form a complex, the pair comprising a first polypeptide comprising at least one binding domain, at least one site susceptible to modification, and a label, whereby the modification of at least one polypeptide is detectable via binding of the binding domain with a second polypeptide; and a second polypeptide which is capable of binding to the first polypeptide, wherein complex formation is detectable via the label.
- the invention also provides an immobilised polypeptide complex, or a constituent polypeptide thereof, which is susceptible to post-translational modification such that modification leads to association or dissociation of the constituent polypeptides from the complex.
- the dissociation or association of the polypeptides in the complex is detectable (further described below).
- the association of the complex is detectable through the interaction of labels placed on two or more polypeptides, this interaction changes depending on whether or not the polypeptides are associated or bound to one another.
- the labels are fluorescent labels
- FRET fluorescence resonance energy transfer
- FRET fluorescence resonance energy transfer
- Methods of detection without use of label are known in the art. These include detection using surface plasmon resonance to detect changes in the mass of the immobilised polypeptide. which would occur if binding of the partner polypeptide increased or decreased. Such measurements may be made for example using a BIAcore machine. Alternatively, mass spectroscopy may be used to detect the presence and mass of a bound species interacting with an immobilised peptide.
- the invention additionally provides a method of screening for a candidate modulator of enzymatic activity of a modification enzyme, the method comprising mixing in an appropriate buffer an appropriate amount of a polypeptide susceptible to modification, wherein the polypeptide binds to at least a second polypeptide, and wherein at least one polypeptide is suitably labelled with detection means for monitoring association/dissociation between the polypeptides; and a sample of material whose enzymatic activity is to be tested; and monitoring the modification of the polypeptide.
- Modulation of the association of the polypeptides to form a complex is indicative of a modulation in the activity of the modification enzyme, and therefore of the activity of the candidate modification enzyme modulator.
- Modulation refers to the capacity to either increase or decrease a measurable signal by at least 10%, 15%, 20%. 25%, 50%>, 100% or more; such increase or decrease is dependent on modification of at least one polypeptide component of a complex.
- biological specimen and “biological sample” refer to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
- body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
- Biological sample further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
- biological sample may refer to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
- organism refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
- a method of the methods described above comprises real-time observation of association of an isolated polypeptide and its binding partner or of an isolated pair of polypeptides.
- the term "real-time” refers to that which is performed contemporaneously with the monitored, measured or observed events and which yields a result of the monitoring, measurement or observation to one who performs it simultaneously, or effectively so, with the occurrence of a monitored, measured or observed event.
- a “real time” assay or measurement contains not only the measured and quantitated result, such as fluorescence, but expresses this in real time, that is, in hours, minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shorter times exceed the instrumentation capability: further, resolution is also limited by the folding and binding kinetics of polypeptides.
- the term "contacting" refers to the act of placing two reagents in such a relationship that they may potentially interact in order to produce a chemical or biological effect.
- the process of contacting involves admixing the reagents at an appropriate concentration in solution or suspension, either in liquid or solid phases, or both, in an appropriate buffer.
- the term "appropriate buffer” refers to a medium which permits activity of the modification enzyme used in an assay of the invention, such as a low-ionic-strength buffer or other biocompatible solution (e.g., water, containing one or more of physiological salt, such as simple saline, and/or a weak buffer, such as Tris or phosphate, or others as described hereinbelow), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate; provided that it is compatible with the binding of the components of the assay of the invention, and with the selected signal employed.
- the buffer advantageously does not include agents which quench fluorescence, if the signal is a fluorescent signal.
- an “appropriate buffer” permits digestion of polypeptides according to the invention and, preferably, inhibits degradation and maintains biological activity or the reaction components. Inhibitors of degradation, such as nuclease inhibitors (e.g., DEPC) are well known in the art.
- an appropriate buffer may comprise a stabilising substance such as glycerol, sucrose or polyethylene glycol.
- the term "appropriate concentration” refers to an amount of reagent (for example, a labelled polypeptide of the invention) which is sufficient for the intended reaction to proceed in a detectable manner.
- reagent for example, a labelled polypeptide of the invention
- an appropriate concentration may be considered to be that concentration at which the label emits a signal within the detection limits of a measuring device used in an assay of the invention. Such an amount is great enough to permit detection of a signal, yet small enough that a change in signal emission is detectable (e.g., such that a signal is below the upper limit of the device).
- the invention relates to a method for detecting or monitoring the activity of a modulator of a modification enzyme, comprising the steps of: a) providing a first polypeptide, and a second polypeptide. wherein i) at least one of the polypeptides is immobilised on a solid support; and ii) the first and second polypeptides are capable of binding to each other in a detectable manner, and modification of one or both of the polypeptides by the modification enzyme results in modulation of the binding of the polypeptides to each other and therefore of the detectable signal; b) allowing the polypeptides to bind to each other and induce a detectable signal; c) contacting the polypeptides with a modification enzyme; d) detecting modulation of the detectable signal as a result of the modulation of the binding of the polypeptides to determine a reference signal modulation; e) contacting the polypeptides with a modification enzyme and a candidate modulator of the modification enzyme; and f) detecting modul
- a “reference signal modulation” is the amount by which a detectable signal is modulated, as defined above, in response to the activity of a modification enzyme in accordance with the invention. For example, therefore, the signal may be modulated, that is increased or decreased, by 10%, 15%, 20%, 25%o, 50%o, 100% or more.
- the reference signal modulation may be calculated at any time, and used as a standard value; it need not be recalculated every time the assay is performed. Comparison of detected signal modulation values with the reference signal modulation preferably manifest themselves as increases or decreases in the percentage modulation with respect to the reference value.
- the assay permits the assessment of the activity of compounds, whether naturally- occurring or synthesised, to modulate the activity of a modification enzyme. It thus permits the use of the invention to detect or monitor processes which rely on modification activity or result in or from modification activity, such as post-translational modifications of proteins.
- the invention preferably relates to a method for detecting or monitoring phosphorylation of a protein, comprising the foregoing steps.
- modulator thus refers to a chemical compound (naturally occurring or synthesised). such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
- Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein.
- the activities (or activity) of a modulator may be known, unknown or partially-known. Such modulators can be screened using the methods described herein.
- test modulator refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator. Usually, various predetermined concentrations are used for screening such as 0.01 ⁇ M, O.l ⁇ M, 1.0 ⁇ M, and 10.0 ⁇ M. as described more fully below.
- Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
- Modulation refers to the capacity to either increase or decease the activity of a modification enzyme by at least 10%, 15%, 20%, 25%, 50%, 100% or more; such increase or decrease may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
- the invention provides the use of an immobilised polypeptide complex according to the preceding aspects of the invention for the detection or monitoring of a protease activity.
- Peptide or protein substrates peculiar to different enzymes can be immobilised to discrete areas of the same physical support, thereby providing a means of examining the activity of a number of enzymes from the same sample in parallel.
- suitable supports include miniature arrays of macromolecules, for example miniature arrays ('chips') bearing polypeptides or peptidomimetics.
- the protein substrate and binding partner can be made using D-amino acids with the exception of the residues which comprise the enzyme's interaction and or modification site, these would be L-amino acids. Most enzymes will not recognise D-amino acid sequences as substrates and thus the likelihood of post-translational modification at unplanned sites is reduced. This refinement is likely to be applicable to assays of the invention.
- Peptide (or protem) sequences each containing at least a single modification site for a different enzyme are immobilised at discrete locations on a physical support.
- a binding partner sequence (or, if necessary, a range of binding partner sequences) is able to associate with these substrate peptides in a modification dependent manner. Association occurs with the unmodified form of the substrate peptide exclusively, but assays with the reverse characteristics can also be configured.
- Exposure of the immobilised polypeptides to an enzyme solution results in the modification of one or more of said polypeptides. Modification of the polypeptide(s) reduces the association with the binding partner polypeptide. This change in association can be measured by any of a variety of techniques as described herein. By placing a range of substrate sequences on the physical support, a range of enzymatic activities or agents can be simultaneously assayed.
- any two amino acid sequences capable of interacting with each other in a modification sensitive manner may be employed.
- the modification site may be natural, i.e. present in at least one of the partner sequences in nature; engineered into the sequence (created by alteration of a natural sequence); or engineered de novo into a wholly synthetic sequence (not known in nature). Binding of these partner structures should occur in one state of modification, but with less stability or not at all in the other.
- sequences of coiled-coil structure can be used in this invention.
- the amino acid sequence of the partner peptide may be varied in order to enhance the stability of binding to the template polypeptide.
- Association of binding partners may be measured by:
- Mass monitor increase in molecular mass of the hybrid species (e.g. use surface plasmon resonance)
- radiolabelled partner e.g. use scintillation proximity assay
- Fluorescence e.g. binding partner is labelled fluorescently: measure fluorescence directly e.g. Use two different fluorophores, one on each of the polypeptides: measure FRET between two fluorescent polypeptides; or a dono ⁇ quencher combination
- Immunology e.g.. use labelled antibody to the binding partner e.g.. use labelled antibody to hapten on binding partner e.g. use labelled antibody to immobilised polypeptide (association of binding partner polypeptide masks antibody binding site on immobilised polypeptide)
- labelled binding partner becomes more immobile upon association: e.g.. use fluorescence anisotropy and/or fluorescence correlation spectroscopy
- Enzymatic reassociation of two parts of an enzyme or photoprotein, or a two- subunit enzyme or photoprotein
- the present invention may be configured in a number of ways. Exemplary embodiments of the invention are set forth below.
- Polypeptides useful in the present invention are capable of associating, either with similar or different polypeptides, to form a polypeptide complex in accordance with the invention.
- Such polypeptides may be naturally-occurring polypeptides, modified naturally-occurring polypeptides, or artificial polypeptides.
- Naturally-occurring polypeptides may be isolated from natural sources or, preferably, synthesised by peptide synthesis or produced using recombinant DNA expression technology.
- Synthetic or partially synthetic polypeptides may be synthesised by peptide synthesis or using recombinant DNA technology and, if necessary, nucleic acid synthesis techniques.
- Various techniques for the synthesis of nucleic acids and peptides are known in the art and may be applied to the present invention.
- labelled polypeptides may be produced via the expression of recombinant nucleic acid molecules comprising an in- frame fusion of sequences encoding a desired polypeptide and a fluorescent protein moiety either in vitro (e.g., using a cell- free transcription/translation system, as described below, or instead using cultured cells transformed or transfected using methods well known in the art) or in vivo, for example in a transgenic animal including, but not limited to, insects, amphibians and mammals.
- a recombinant nucleic acid molecule of use in the invention may be constructed and expressed by molecular methods well known in the art, and may additionally comprise sequences including, but not limited to, those which encode a tag (e.g., a histidine tag) to enable easy purification, a secretion signal, a nuclear localisation signal or other primary sequence signal capable of targeting the construct to a particular cellular location, if it is so desired.
- Assays may be configured such that a number of modifications can be monitored using unique, modified polypeptides peculiar to each assay and one common polypeptide, which is important in the automation of assays.
- polypeptides useful in the present invention may be defined as follows. Firstly, the polypeptide requires a binding site which will permit it to bind to other polypeptides to form a complex.
- Polypeptides are known to be able to associate in a number of ways, and domains which mediate polypeptide association are known in the art. For example, coiled coils, acid patches, zinc fingers, calcium hands, WD40 motifs, SH2/SH3 domains and leucine zippers are all polypeptide domains known to mediate protein-protein interactions, as are other domains known to those skilled in the art.
- Some examples of polypeptide interacting domains which may be suitable for use in this invention are presented in the following table:
- ZIP contains more than one protein binding motif (YXDED motif, ZZ zinc finger) and is known to bind to several proteins other than PKC ⁇ (including p62 and EBLAP) and also to self-associate (this self association is in competition with PKC ⁇ binding). These multiple interactions should be considered when designing an assay according to the present invention.
- the coiled-coil domain is structurally conserved among many proteins that interact to form homo- or heterodimeric oligomers.
- the leucine zipper provides an example of one such protein structural motif. It is found in, among other examples, a nuclear protein that functions as a transcriptional activator of a family of genes involved in the General Control of Nitrogen (GCN4) metabolism in S. cerevisiae. The protein is able to dimerise and bind promoter sequences containing the recognition sequence for GCN4, thereby activating transcription in times of nitrogen deprivation.
- hydrophobic residues in particular alanine, isoleucine, leucine, methionine or valine
- hydrophobic residues are present at the 'a' and 'd' positions within a heptad when the amino acids are identified as positions a,b,c,d,e,f and g by order of sequence.
- spacing of hydrophobic residues in patterns of 3,4,4 and 3,4,3 (hendecad repeat) have recently been reported (Hicks et al, 1997, Folding and Design., 2: 149-158) and are compatible with the coiled-coil structure, (ii)
- coiled-coil helical bundles have between 2 and 5 helices which are offset at roughly 20° to adjacent strands with the hydrophobic sidechains interdigitating in the interface between helices in what is termed the "knobs into holes" packing (Crick, 1953, Acta. Crystallogr., 6: 689-697).
- Natural and non-natural coiled-coils can have parallel and/or antiparallel helices. Both homotypic (multiple strands of identical sequence) and hetero
- Leucine zipper sequences conform to the coiled- coil rules above and typically have leucine residues at the 'd' position of the canonical heptad repeat. These leucine residues represent a single face of the helix. Interdigitating with these leucine residues are other hydrophobic amino acids, frequently valine, isoleucine or leucine residues. The combination of these residues forms a continuous hydrophobic face which associates with an equivalent region in an associating subunit. Alternatively the hydrophobic face can be discontinuous due to interruptions in the heptad repeat sequence. This, however, does not interfere with the ability of these coiled-coils to interact.
- the stability of the dimer thus formed is conferred by the hydrophobic interactions between the leucine residues and the interdigitating hydrophobic residues. Hydrogen bonds that form between residues present on the two interacting helices, particularly at the e and g positions, also contribute to the stability of the dimer.
- the coiled-coil domain of GCN4 has been shown to dimerise as an isolated peptide (Gonzalez et al., 1996, Nature Structural Biology. 3: 1011-1018).
- thermophilus DLEALLALDREVQELKKRLQEVQTERNQVAKRV
- the binding domains may be similar or different, i.e. homomultimeric or heteromultimeric.
- the rules for the design of hetero-multimeric coiled coils are well detailed in the literature (including Peptide 'Velcro': design of a heterodimeric coiled coil, O'Shea, E.K., Lumb, K.J. & Kim, P.S., Current Biology (1993) 3 658-667; A designed heterotrimeric coiled coil, Nautiyal, S., Woolfson, D.N., King, D.S.
- polypeptides may also associate via interactions, not necessarily involving canonical domains such as coiled-coils, which may be specific to the polypeptides in question.
- one or more polypeptides in each multimer may comprise a label.
- Suitable fluorescent labels include fluorophores and fluorescent proteins.
- fluorophore and “fluorochrome” refer interchangeably to a molecule which is capable of absorbing energy at a wavelength range and releasing energy at a wavelength range other than the absorbance range.
- excitation wavelength refers to the range of wavelengths at which a fluorophore absorbs energy.
- emission wavelength refers to the range of wavelength that the fluorophore releases energy or fluoresces.
- fluorescent proteins which vary among themselves in excitation and emission maxima are listed in Table 1 of WO 97/28261 (incorporated herein by reference). These (each followed by [excitation max./emission max.] wavelengths expressed in nanometers) include wild-type Green Fluorescent Protein [395(475)/508] and the cloned mutant of Green Fluorescent Protein variants P4 [383/447], P4-3 [381/445], W7 [433(453)7475(501)], W2 [432(453)7480], S65T [489/511], P4-1 [504(396)/480], S65A [471/504], S65C [479/507], S65L [484/510], Y66F [360/442], Y66W [458/480], IOc [513/527], W1B [432(453)/476(503)], Emerald [487/508] and Sapphire [395/51 1]. This list is not exhaustive of fluorescent proteins known in the art
- a number of parameters of fluorescence output are envisaged including 1) measuring fluorescence emitted at the emission wavelength of the acceptor (A) and donor (D) and determining the extent of energy transfer by the ratio of their emission amplitudes; 2) measuring the fluorescence lifetime of D;
- Labels may be attached in a number of ways, such as by direct labelling at suitable amino acids, such as cysteines or lysines, with chemical labels, or by fusion with a polypeptide label such as a fluorescent polypeptide or photoprotein subunit or fragment. Techniques for labelling polypeptides are generally known in the art and may be applied to the present invention.
- one or more of the polypeptides in each complex according to the invention may be susceptible to modification.
- susceptibility to modification indicates that the polypeptide may be subjected to proteolytic degradation under the appropriate conditions, which in a preferred embodiment means that the polypeptide is cleaved by a protease at a recognition site for the protease enzyme.
- the polypeptide may be susceptible to digestion by an exoprotease, from the N or C terminus.
- the polypeptide may be susceptible to acylation under the appropriate conditions.
- the polypeptide may be susceptible to phosphorylation under appropriate conditions.
- Peptides may be rendered susceptible to modification by inclusion within the peptide sequence of a recognition site for a modification enzyme. This may be performed using peptide synthesis techniques as described above.
- Polypeptides according to the invention should be constructed such that the protease cleavable site is positioned such that cleavage thereof disrupts binding of the polypeptide in the context of the multimer.
- polypeptides which have been subjected to protease cleavage should dissociate from the multimer.
- the protease does not cleave the polypeptide in such a manner that the label becomes detached therefrom without the binding abilities thereof being disrupted.
- Location of the protease cleavable site may be determined empirically. As a guide, however, the site should be placed within or proximal to the binding domain which is responsible for the multimerisation of the polypeptide.
- Lumb et al Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4, Lumb, K.J., Carr, CM. & Kim, P.S., Biochemistry (1994) 33 7361-7367
- GCN4-p 1 the loss of ten residues from the N-terminus or seven from the N- and six from the C-terminus is sufficient to destabilise the coiled coil peptide sequence known as GCN4-p 1
- the leucine zipper is a coiled coil, O'Shea, E.K., Rutkiowski, R. & Kim, P.S. (1989) Science 243 538-542).
- Su et al provide data indicating that there is a sha ⁇ decrease in the stability of a designed coiled coil with a decrease in chain length from 23 to 19 residues (Su, J.Y., Hodges, R.S. & Kay, CM., Biochemistry (1994) 33 15501-15510). Accordingly, cleavage sites may positioned such that the coiled coil is disrupted to an extent that it is no longer capable of directing multimerisation.
- the cleavage sites of a number of proteases are known in the art, and set forth in Table 3.
- D-isomers of amino acids in the construction of the polypeptide.
- D-amino acids are resistant to protease digestion, and a polypeptide constructed of D-amino acids will withstand proteolytic attack.
- use of D-amino acids does not interfere with the protein- protein interactions involved in multimerisation, such as the interaction of protein binding domains, especially coiled-coil domains, provided that D amino acids are employed in both members of a binding pair.
- the D-amino acid constructions of the polypeptides of the invention contain one or more parts constructed of L-amino acids, or otherwise rendered susceptible to proteolytic digestion.
- coiled coils constructed of D-amino acids preferably comprise inserts, constructed wholly or partly of L-amino acids, which contain the protease cleavage site.
- the L-amino acid insert may be of any size, and may be positioned between coiled coil repeats, or between residues of the coiled coil. Preferred are insertions at positions b-c, e- f and f-g.
- the insert is covalently attached to the coiled coil, through a covalent linkage to the backbone or through a sidechain.
- the insert may comprise only a cleavage site, or an entire polypeptide. Functionally, the insert is sufficiently flexible to permit the coiled coil to bind to its target efficiently when the insert is intact.
- the insert may comprise a flexible linker, such as a gly- gly linker. Molecules comprising D-amino acids are advantageously employed in in vitro assays.
- Inserts as described above may be employed in D-amino acid coiled coils, in conventional L-amino acid coiled coils, or in coiled coils which are partially D and partially L in construction.
- a coiled coil may be constructed such that it consist of L- amino acids on one side of the insert, and D-amino acids on the other side thereof.
- signal useful in the present invention may be generated by a number of different labels.
- FRET energy transfer
- FRET is detectable when two fluorescent labels which fluoresce at different frequencies are sufficiently close to each other that energy is able to be transferred from one label to the other.
- FRET is widely known in the art (for a review, see Matyus, 1992, J. Photochem. Photobiol. B: Biol, 12: 323-337, which is herein inco ⁇ orated by reference).
- FRET is a radiationless process in which energy is transferred from an excited donor molecule to an acceptor molecule; the efficiency of this transfer is dependent upon the distance between the donor an acceptor molecules, as described below. Since the rate of energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, the energy transfer efficiency is extremely sensitive to distance changes. Energy transfer is said to occur with detectable efficiency in the 1-10 nm distance range, but is typically 4-6 nm for favourable pairs of donor and acceptor.
- Fluorescenceless energy transfer is based on the biophysical properties of fluorophores. These principles are reviewed elsewhere (Lakowicz, 1983, Principles of Fluorescence Spectroscop y , Plenum Press, New York; Jovin and Jovin, 1989, Cell Structure and Function bv Microspectrofluorometry, eds. E. Kohen and J.G. Hirschberg, Academic Press, both of which are inco ⁇ orated herein by reference). Briefly, a fluorophore absorbs light energy at a characteristic wavelength. This wavelength is also known as the excitation wavelength. The energy absorbed by a fluorochrome is subsequently released through various pathways, one being emission of photons to produce fluorescence.
- the wavelength of light being emitted is known as the emission wavelength and is an inherent characteristic of a particular fluorophore. Radiationless energy transfer is the quantum- mechanical process by which the energy of the excited state of one fluorophore is transferred without actual photon emission to a second fluorophore. That energy may then be subsequently released at the emission wavelength of the second fluorophore.
- the first fluorophore is generally termed the donor (D) and has an excited state of higher energy than that of the second fluorophore, termed the acceptor (A).
- the essential features of the process are that the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, and that the donor and acceptor be sufficiently close.
- the distance over which radiationless energy transfer is effective depends on many factors including the fluorescence quantum efficiency of the donor, the extinction coefficient of the acceptor, the degree of overlap of their respective spectra, the refractive index of the medium, and the relative orientation of the transition moments of the two fluorophores.
- the distance between D and A must be sufficiently small to allow the radiationless transfer of energy between the fluorophores.
- FRET may be performed using proteins labelled by methods known in the art.
- two coiled-coil domains comprised either by the same or by different polypeptide molecules are differentially labelled, one with a donor and the other with an acceptor moiety, and differences in fluorescence between a test assay, comprising a protein modifying enzyme, and a control, in which the modifying enzyme is absent, are measured using a fluorimeter or laser-scanning microscope.
- excitation/detection means can be augmented by the inco ⁇ oration of photomultiplier means to enhance detection sensitivity.
- the differential labels may comprise either two different fluorescent moieties (e.g., fluorescent proteins as described below or the fluorophores rhodamine, fluorescein, SPQ, and others as are known in the art) or a fluorescent moiety and a molecule known to quench its signal.
- fluorescent moieties e.g., fluorescent proteins as described below or the fluorophores rhodamine, fluorescein, SPQ, and others as are known in the art
- the fluorescent labels are chosen such that the excitation spectrum of one of the labels (the acceptor label) overlaps with the emission spectrum of the excited fluorescent label (the donor label).
- the donor label is excited by light of appropriate intensity within the donor's excitation spectrum.
- the donor then emits some of the absorbed energy as fluorescent light and dissipates some of the energy by FRET to the acceptor fluorescent label.
- the fluorescent energy it produces is quenched by the acceptor fluorescent label.
- FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor, reduction in the lifetime of its excited state, and re- emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor. When the donor and acceptor labels become spatially separated, FRET is diminished or eliminated.
- Two distinct polypeptides each comprising a coiled-coil may be differentially labelled with the donor and acceptor fluorescent protein moieties, respectively.
- polypeptides are assayed for association using fluorescent protein moiety labels according to the invention may be briefly summarised as follows: Of two polypeptides which associate into a complex according to the present invention, one is labelled with a green fluorescent protein, while the other is preferably labelled with a red or, alternatively, a blue fluorescent protein.
- Useful dono ⁇ acceptor pairs of fluorescent proteins include, but are not limited to:
- Donor S72A, K79R, Y145F, M153A and T203I (excitation 395nm; emission 511)
- Acceptor S659, S72A, K79R and T203Y (wavelengths not noted), or T203Y/S65G, V68L, Q69K or S72A (excitation 515nm; emission 527nm).
- P4-3 shown in Table 1 of WO 97/28261
- S65C also of Table 1 of WO 97/28261
- the polypeptides are exposed to light at, for example, 368 nm, a wavelength that is near the excitation maximum of P4-3. This wavelength excites S65C only minimally.
- some portion of the energy absorbed by the blue fluorescent protein moiety is transferred to the acceptor moiety through FRET if the two polypeptides are in close association.
- the blue fluorescent light emitted by the blue fluorescent protein is less bright than would be expected if the blue fluorescent protein existed in isolation.
- the acceptor moiety (S65C) may re-emit the energy at longer wavelength, in this case, green fluorescent light.
- polypeptides physically separate, accordingly inhibiting FRET.
- Such a system is useful, for example to monitor the activity of proteolytic enzymes that cleave polypeptides to which the fluorescent labels are fused as well as the activity of modulators or candidate modulators of those enzymes.
- the invention contemplates assays in which the amount or activity of a modification agent in a sample is determined by contacting the sample with a pair of polypeptides differentially labelled with fluorescent proteins, as described above, and measuring changes in fluorescence of the donor moiety, the acceptor moiety or the relative fluorescence of both.
- Advantages of fluorescent polypeptides constructed as fusions with fluorescent proteins include the greater extinction coefficient and quantum yield of many of these proteins compared with those of the Edans fluorophore.
- the acceptor in such a construct or pair of constructs is, itself, a fluorophore rather than a non-fluorescent quencher like Dabcyl.
- the enzyme's substrate i.e., the unmodified polypeptide of the construct(s) and products (i.e., the polypeptides after modification) are both fluorescent but with different fluorescent characteristics.
- the substrate and modified products exhibit different ratios between the amount of light emitted by the donor and acceptor labels. Therefore, the ratio between the two fluorescence emission values measures the degree of conversion of substrate to products, independent of the absolute amount of either, the optical thickness of the sample, the brightness of the excitation lamp, the sensitivity of the detector, etc.
- Aequorea-de ⁇ vs ⁇ or related fluorescent protein moieties tend to be resistant to degradation, e.g. by proteases. Therefore, they are likely to retain their fluorescent properties throughout the course of an experiment.
- a signal may be generated by using a redox enzyme as a label.
- a redox enzyme as a label.
- Such systems are based on glucose oxidase (GOX) sensor technology.
- GOX glucose oxidase
- a number of glucose sensors have been produced to determine the concentration of glucose in a biological fluid (see US patents 5605152, 5849174, 5215887, 5082786, 577967, 5755953, 5225064 and 5682884; inco ⁇ orated herein by reference).
- the invention may be configured to exploit first generation GOX technology, which is oxygen-dependent:
- the glucose concentration can be coupled to a means of detection, including the following preferred techniques.
- the first is to measure the decrease in pH caused by the production of gluconic acid.
- GOX is immobilised on an ion sensing device. On dipping into the sample fluid any glucose present leads to catalytic turnover and the production of gluconic acid.
- the second method couples the turnover of GOX to that of horseradish peroxidase (HRP).
- HRP catalyses the conversion of hydrogen peroxide to water, equation (iii).
- the redox active dye 0-dianisidine reduces H O . Formation of the radical cation of the dye reflects the catalytic activity of GOX and is monitored spectrophotometrically at 440nm.
- second generation GOX technology is employed. This has the advantage of producing an electrical signal in response to stimulus, which is easily assayed.
- the device described is a small, two electrode strip.
- One electrode is a reference electrode consisting of Ag and/or AgCl.
- the other is a carbon electrode.
- 0 2 is replaced by a redox active mediator such as ferricyanide, [Fe(CN) 6 ] 3 ⁇ or a ferrocene derivative (l,l '-dimethylferrocene).
- the mediator and GOX are screen printed onto the carbon electrode.
- the sample fluid is placed over both electrodes. GOX will turn over on any glucose present (iv).
- the mediator acts to transfer electrons from GOX to the electrode surface (v).
- the potential difference produced is measured by a voltmeter or similar device.
- a polypeptide according to the invention is immobilised on the surface of a carbon electrode in a standard GOX assay.
- the mediator ferrerricyanide or ferrocene
- the sample fluid drop is placed on the electrodes.
- a GOX-fusion binding partner peptide may be included in this screen print formulation or added after the sample. Either way the GOX-fusion binding partner binds to the immobilised polypeptide positioning GOX is at the surface of the electrode. Providing glucose is present, GOX will turn over passing electrons to the electrode via the mediator. Thus an output is provided conditional on binding of the binding partner polypeptide to the immobilised polypeptide.
- the potential difference generated is measured with a voltmeter or similar instrument.
- GOX is removed from the surface of the electrode and the circuit is broken.
- the glucose assay is advantageously configured such that it operates efficiently when the binding partner polypeptide, and so GOX, are held close to the electrode by the immobilised polypeptide.
- This system couples the glucose assay to polypeptide modification events and uses it as a signal. For example, where coiled-coil dimers are disrupted by phosphorylation events, the signal is indicative of the phosphorylation state of the polypeptides. Prior to phosphorylation GOX turns over on glucose and a potential difference is observed. When phosphorylation of the peptides occurs, the dimer dissociates and enzymatic turnover is impaired, reducing the potential difference. Such a system is equally applicable to other modifications, such as proteolysis.
- the system may be extended using a wide range of redox proteins and substrates in place of the glucose oxidase system. These include:
- the invention permits the design of a multiple assay system.
- Each assay produces an electrical signal allowing monitoring of the enzymatic reaction in each case.
- a series of assays may be performed on one biological sample.
- a range of substrates may be included to assay for the substrate specificity of one enzyme.
- a polypeptide according to the invention is immobilised on the electrode surface.
- a binding partner polypeptide is fused to the enzyme of interest and may be included in the screen print formulation or added to the sample fluid.
- Digestion of polypeptides according to the invention with proteolytic enzymes may be carried out according to techniques and procedures known in the art.
- appropriate amounts of polypeptides according to the invention are incubated in appropriate conditions, for example with an appropriate buffer.
- the term "appropriate buffer” refers to a medium which permits activity of the protease enzyme used in an assay of the invention, and is typically a low-ionic- strength buffer or other biocompatible solution (e.g., water, containing one or more of physiological salt, such as simple saline, and/or a weak buffer, such as Tris or phosphate, or others as described hereinbelow), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate; provided that it is compatible with the binding of the components of the assay of the invention, and with the selected signal employed.
- the buffer advantageously does not include agents which quench fluorescence, if the signal is a fluorescent signal.
- an “appropriate buffer” permits modification of polypeptides according to the invention and, preferably, inhibits degradation and maintains biological activity or the reaction components. Inhibitors of degradation, such as nuclease inhibitors (e.g., DEPC) are well known in the art.
- an appropriate buffer may comprise a stabilising substance such as glycerol, sucrose or polyethylene glycol.
- the term "appropriate amounts of polypeptides” refers to an amount of labelled polypeptides of the invention which emit a signal within the detection limits of a measuring device used in an assay of the invention. Such an amount is great enough to permit detection of a signal, yet small enough that a change in signal emission is detectable (e.g., such that a signal is below the upper limit of the device).
- the assay may be configured to use polypeptides, which are cleavable by a protease at a specific protease cleavable site.
- This assay requires the formation of an oligomer of binding partners containing a proteolytic site or with a proteolytic site engineered therein which are labelled with fluorophores appropriate for FRET (or some other appropriate means of detection).
- the coiled coil provides a good example of such an oligomer.
- the sequence of the coiled coil part of the peptides in this reporter is short, such that it contains at least two, and preferably three, four or five, heptad structures. Within this heptad structure an amino acid sequence recognised as a cleavage site for a protease is included. Cleavage of the peptide by the protease will reduce the length of the continuous coiled coil sequence, and destabilise oligomer formation.
- the fluorophores (FI, F2; where FI is the donor fluorophore and F2 the acceptor) should be positioned such that they are covalently attached to separate peptides in the oligomeric complex at positions which are close in space in this complex but are not attached to a residue which affects enzyme processing of the substrate (see Figure 18A) This illustrates an assay based on a homo-oligomeric assembly of peptides.
- the assay may, however, also be configured as a heteromultimeric assay in which one or more of the polypeptides comprises a protease cleavage site ( Figure 18B).
- the assay may be modified with respect to the diagram shown in Figure 18B to permit proteolytic degradation of more than one polypeptide in the complex.
- one of the advantages of heteromultimeric assays is that only a single polypeptide is digested.
- the invention may be configured to monitor the degradation of polypeptides, especially naturally-occuring polypeptides, as a result of proteolytic degradation.
- a polypeptide may be produced as a fusion with a further polypeptide which is to be assayed for proteolytic degradation.
- Protease enzymes which degrade the further polypeptide will also degrade the polypeptide of the invention, giving rise to complex dissociation.
- Single-polypeptide reagents may be configured in a number of ways, depending on the location of the labels with respect to the binding domains on the polypeptide.
- the labels are positioned N- and C-terminal to the respective binding domains (see Figure 19B).
- Polypeptides may be produced which associate in a phosphorylation-dependent manner. This may mean that association only occurs if one or more of the polypeptides is phosphorylated at one or more sites. Alternatively, this may mean that the polypeptides only associate if they are in the unphosphorylated state.
- the polypeptide which is susceptible to phosphorylation is immobilised and associates with a second polypeptide in the unphosphorylated state.
- the association is measurably reduced.
- the assay may be configured such that the polypeptides associate only in the phosphorylated state.
- Interaction or association of polypeptides may be measured by any suitable means as set out in this application. It will be recognised by those skilled in the art that some means of detection allow the monitoring of multiple pairs of interacting polypeptides by choosing the fluorophore labels in such a way that different pairs of polypeptides are labelled with different colour fluorophores (i.e. FRET pairs which are configured such that their abso ⁇ tion and/or emission spectra are different from one another).
- FRET pairs which are configured such that their abso ⁇ tion and/or emission spectra are different from one another.
- two such pairs may have similar abso ⁇ tion wavelengths, with different emission wavelengths, or may have different abso ⁇ tion wavelengths with similar emission wavelengths, or may differ from one another in both their abso ⁇ tion and emission wavelengths. It will be understood that for the practice of the invention in this manner, the only requirement is that the fluorophores are arranged in such a way that FRET pairs may be separately monitored or otherwise distinguished.
- Molecules according to the invention are advantageously produced in insect cell systems.
- Insect cells suitable for use in the method of the invention include, in principle, any lepidopteran cell which is capable of being transformed with an expression vector and expressing heterologous proteins encoded thereby.
- Sf cell lines such as the Spodoptera frugiperda cell line IPBL-SF-21 AE (Vaughn et al., (1977) In Vitro, 13, 213-217) is preferred.
- the derivative cell line Sf9 is particularly preferred.
- other cell lines such as Tricoplusia ni 368 (Kurstack and Marmorosch, (1976) Invertebrate Tissue Culture Applications in Medicine, Biology and Agriculture.
- insect cell lines as well as other insect cell lines suitable for use in the invention, are commercially available (e.g. from Stratagene, La Jolla, CA, USA).
- the invention also comprises the expression of polypeptides in whole insect organisms.
- virus vectors such as baculovirus allows infection of entire insects, which are in some ways easier to grow than cultured cells as they have fewer requirements for special growth conditions.
- the protein can be extracted from the insects according to conventional extraction techniques.
- Expression vectors suitable for use in the invention include all vectors which are capable of expressing foreign proteins in insect cell lines.
- vectors which are useful in mammalian and other eukaryotic cells are also applicable to insect cell culture.
- Baculovirus vectors, specifically intended for insect cell culture are especially preferred and are widely obtainable commercially (e.g. from Invitrogen and Clontech).
- Other virus vectors capable of infecting insect cells are known, such as Sindbis virus (Hahn et al., (1992) PNAS (USA) 89, 2679-2683).
- the baculovirus vector of choice (reviewed by Miller (1988) Ann. Rev. Microbiol. 42, 177-199) is Autographa californica multiple nuclear polyhedrosis virus, AcMNPV.
- the heterologous gene replaces at least in part the polyhedrin gene of AcMNPV, since polyhedrin is not required for virus production.
- a transfer vector is advantageously used. Transfer vectors are prepared in E. coli hosts and the DNA insert is then transferred to AcMNPV by a process of homologous recombination.
- molecules according to the invention may be expressed in bacterial, lower eukaryote or mammalian cell systems, or in transgenic animals.
- vector or plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles is well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function and the host cell for which it is compatible.
- the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
- Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells.
- this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
- origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
- the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells.
- the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.
- Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another class of organisms for expression.
- a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome.
- DNA may also be replicated by insertion into the host genome. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.
- an expression and cloning vector may contain a selection gene also referred to as selectable marker.
- This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
- Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.
- Selectable markers which may be used in fungal cells include wild- type genes which complement auxotrophic defects in for example the Uracil (e.g... URA3 gene), Lysine (e.g.. LYS2 gene), Adenine (e.g.. ADE2 gene), Methionine (e.g.. MET3 gene), Histidine (e.g.. HIS3 gene), Tryptophan (e.g.. TRP1 gene), Leucine (e.g... LEU2 gene) or other metabolic pathways.
- Uracil e.g... URA3 gene
- Lysine e.g.. LYS2 gene
- Adenine e.g.. ADE2 gene
- Methionine e.g.. MET3 gene
- Histidine e.g.. HIS3 gene
- Tryptophan e.g... TRP1 gene
- Leucine e.g.. LEU2 gene
- Examples of these include; 5-fluoro-orotic acid, which is converted to a toxic compound by the action of the URA3 gene product; ⁇ -amino-adipic acid, which is converted to a toxic compound by the LYS2 gene product; allyl alcohol, which is converted to a toxic compound by alcohol dehydrogenase activity as encoded by the ADH genes, or any other suitable selective regime known to those skilled in the art.
- Other selective markers are based on the expression of a gene in a fungus such as yeast which overcomes the metabolic arrest induced by, or toxicity of, a chemical entity which may be added to the growth medium or otherwise presented to the cells.
- Examples of these may include the KAN gene(s) which confer resistance to antibiotics such as G-418, the HIS3 gene which confers resistance to 3- amino-triazole, or the ADH2 gene which can confer resistance to heavy metal ions such as cadmium, or any other suitable genes which confer resistance to toxic or growth arresting regimes.
- an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript ⁇ vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
- Suitable selectable markers for mammalian cells are those that enable the identification of cells which have taken up vectors according to the invention, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin.
- DHFR dihydrofolate reductase
- methotrexate resistance thymidine kinase
- genes conferring resistance to G418 or hygromycin conferring resistance to G418 or hygromycin.
- selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes a polypeptide according to the invention.
- Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the chromosomes of recombinant cells. Increased quantities of desired polypeptide are usually synthesised from thus amplified DNA.
- Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to a coding sequence. Such a promoter may be inducible or constitutive.
- the promoters are operably linked to DNA encoding a polypeptide according to the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native promoter sequence associated with the polypeptide in question, where this is naturally occurring, and many heterologous promoters may be used to direct amplification and/or expression of nucleic acids.
- the term "operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
- a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
- Promoters suitable for use with prokaryotic hosts include, for example, the ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (t ⁇ ) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding a polypeptide according to the invention, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine- Delgarno sequence operably linked to the DNA encoding a polypeptide according to the invention.
- Preferred expression vectors are bacterial expression vectors which comprise a promoter of a bacteriophage such as phagex or T7 which is capable of functioning in the bacteria.
- the nucleic acid encoding the fusion protein may be transcribed from the vector by T7 RNA polymerase (Studier et al, Methods in Enzymol. 185; 60-89, 1990).
- T7 RNA polymerase In the E. coli BL21(DE3) host strain, used in conjunction with pET vectors, the T7 RNA polymerase is produced from the ⁇ -lysogen DE3 in the host bacterium, and its expression is under the control of the IPTG inducible lac UV5 promoter. This system has been employed successfully for over-production of many proteins.
- the polymerase gene may be introduced on a lambda phage by infection with an int- phage such as the CE6 phage which is commercially available (Novagen, Madison, USA), other vectors include vectors containing the lambda PL promoter such as PLEX (Invitrogen, NL) , vectors containing the trc promoters such as pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE) , or vectors containing the tac promoter such as pKK223-3 (Pharmacia Biotech) or PMAL (new England Biolabs, MA, USA).
- PLEX Invitrogen, NL
- vectors containing the trc promoters such as pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE)
- vectors containing the tac promoter such as pKK223-3 (Pharmaci
- nucleic acids encoding polypeptides according to the invention preferably include a secretion sequence in order to facilitate secretion of the polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.
- the peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.
- Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene.
- the S. pombe nmt 1 gene or a promoter from the TATA binding protein (TBP) gene can be used.
- TBP TATA binding protein
- hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05-GAP hybrid promoter).
- a suitable constitutive PHO5 promoter is e.g.
- PH05 a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide -173 and ending at nucleotide -9 of the PH05 gene.
- UAS upstream regulatory elements
- Gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated the naturally occurring sequence encoding the polypeptide at issue, provided such promoters are compatible with the host cell systems.
- viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the act
- Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.
- a eukaryotic expression vector encoding a polypeptide according to the invention may comprise a locus control region (LCR).
- LCRs are capable of directing high-level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the gene is to be expressed in the context of a permanently-transfected eukaryotic cell line in which chromosomal integration of the vector has occurred, in vectors designed for gene therapy applications or in transgenic animals.
- Eukaryotic expression vectors will also contain sequences necessary for the termination of transcription and for stabilising the mRNA. Such sequences are commonly available from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide according to the invention.
- An expression vector includes any vector capable of expressing nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs.
- an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA.
- Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
- DNAs encoding a polypeptide according to the invention may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) Nucl. Acid. Res. 17, 6418).
- a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) Nucl. Acid. Res. 17, 6418).
- Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of polypeptides according to the invention.
- transient expression systems are useful e.g. for identifying mutants of polypeptides according to the invention, to identify potential phosphorylation sites, or to characterise functional domains of the protein.
- Plasmids according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe which may be based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
- cells containing the above-described nucleic acids there are provided cells containing the above-described nucleic acids.
- host cells such as prokaryote, yeast and higher eukaryote cells may be used for replicating DNA and producing polypeptides according to the invention.
- Suitable prokaryotes include eubacteria, such as Gram- negative or Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5 ⁇ and HB101, or Bacilli.
- Further hosts suitable for polypeptides according to the invention encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae.
- Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells, including human cells, or nucleated cells from other multicellular organisms.
- mammalian host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells.
- the host cells referred to in this disclosure comprise cells in in vitro culture as well as cells that are within a host animal.
- DNA may be stably inco ⁇ orated into cells or may be transiently expressed using methods known in the art.
- Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.
- the cells should be transfected with a sufficient amount of polypeptides according to the invention-encoding nucleic acid to form polypeptides according to the invention.
- the precise amounts of DNA encoding polypeptides according to the invention may be empirically determined and optimised for a particular cell and assay.
- Host cells are transfected or, preferably, transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
- Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.
- Inco ⁇ oration of cloned DNA into a suitable expression vector transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).
- Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby polypeptides according to the invention encoded by the DNA is expressed.
- the composition of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also commercially available.
- Polypeptides according to the invention may moreover by prepared by chemical synthesis, using standard equipment and techniques. Exemplary techniques are discussed herein, and are well known in the art. They include Fmoc and Tboc chemistry according to the methods of Atherton, E., Logan, C.J., & Sheppard (1981), J. Chem. Soc. 1981 538-546 and Perkin. I. & Merrifield R.B. (1963) J. Am. Chem. Soc. 85, 2149-2154.
- the labelling reagent may be a group conferring the desired property such as fluorescence, and preferably includes a group involved in the conjugation of said label to the target polypeptide.
- the most commonly used functional groups in this context are those which react with amines by either acylation or alkylation. These include isothiocyanates, isocyanates. acyl azides, NHS esters and many others. Also common is the use of thiol- directed groups such as haloacetates and maleimides. These label primarily at the free sulfhydryl group of cysteine residues.
- Buffer/ionic strength - the incubation solution may comprise an appropriate buffer (as defined above) of an ionic strength suitable for the formation of a folded reporter molecule structure (likely to be 0-150 mM. of a suitable salt).
- Concentration - peptides may be present at a concentration sufficiently high to allow formation of the folded reporter molecule and detection of the assay output (within the limits of instrumentation) but not so high that the detector is saturated. Temperature - the temperature selected will preferably be suitable for both formation of the reporter multimer and also activity of any biological agents included in the assay (for example 4-40°C).
- each of these parameters is empirically determined as the behaviour of the reporter molecule may be sequence dependent.
- polypeptides of the invention comprise two or more parts of an enzymatic activity
- the association of the polypeptides may be measured by monitoring the modulation of this enzymatic activity.
- This monitoring may be via some kind of biosensor, for example using Glucose oxidase as the enzyme as part of a miniature electrical sensor circuit.
- the polypeptides of the invention may be designed in such a way that, when they associate, they reform an inhibitor of an enzymatic activity.
- the association of the polypeptides may be measured by monitoring the modulation of said enzymatic activity.
- PKC activity may be monitored, and this activity may be modulated by association of polypeptides according to the invention reforming PKC inhibitor (PKCi).
- PKCi PKC inhibitor
- the enzymes described can be purified from natural sources or from cells/microorganisms engineered to heterologously express these enzymes. All enzymes used to illustrate this invention are commercially available; details of cut and modification sites are shown in Table 3. The recombinant forms of these enzymes are sometimes expressed with extra tags (such as the 6-histidine extension) for the pu ⁇ ose of purification.
- Peptides can be synthesised from the heterologous expression of cDNA sequences for domains of interest can be modified to include sequences for modification as appropriate, or synthetic gene of the same.
- Expression can be in prokaryotic or eukaryotic cells using a variety of plasmid vectors capable of instructing heterologous expression. Purification of these products is achieved by destruction of the cells (e.g. French Press) and chromatographic purification of the products. This latter procedure can be simplified by the inclusion of an affinity purification tag at one extreme of the peptide, separated from the peptide by a protease cleavage site (other than that of interest) if necessary.
- Heterologous expression of the peptides as described above can also be adapted to facilitate in vitro or in vivo assay of modification events.
- the cDNA or synthetic gene used codes for the peptide to be expressed fused to a variant of GFP, at the N or C terminus.
- Two plasmids or a plasmid carrying two genes can be introduced such that two peptides are expressed, capable of forming a dimer and fused with different GFP variants capable of FRET.
- a dimer forms and FRET is observed. Modification events destroy the dimer and FRET is lost.
- the expression of the peptides in the cell to be investigated removes the need for micro injection of the reporter peptides.
- Labelling of peptides Peptides are labelled with thiol reactive or primary amine reactive derivatives of fluorescein and tetramethylrhodamine or other suitable FRET partners (see table 2; Molecular Probes, Eugene, OR, USA) using procedures described by Hermanson, G.T (1996) Bioconjugate Techniques, Academic Press. London.
- Fluorescent (labelled) peptides are subsequently separated from the non-labelled peptides and the unreacted fluorophores by gel filtration or reverse phase chromatography.
- Chymotrypsin - Chymotrypsin is a small proteolytic enzyme (MW 21000 Da) found in the gastrointestinal tract. It catalyses the hydrolysis of digestive proteins to large peptides. The cleavage of peptides is on the C terminal side of Y-X, F-X, W-X, L-X, M-X, A-X, D- X, E-X,.
- the peptide structures of this invention are adapted to provide a label molecule on the template which carries the modification site as well as the tag for immobilisation onto a physical support. The reporter molecule enables monitoring of cleavage by chymotrypsin.
- HHHHHHGGIAQLEQEIAQLEQENAYLEQEIAQLEQEIAKLEQE Y represents the cleavage site for chymotrypsin with the cut occurring between Y and L residues
- K represents the fluorophore attachment site and the 6 His residues are used for immobilisation of the polypeptide.
- Binding partner polypeptide IAQLKQKIAQLKQKNAQLKQKIAQLKQKICQLKQK C represents the label attachment site.
- Lyophilised chymotrypsin is dissolved in 1 mM HCI. Peptides (0.001-100 ⁇ M) are cleaved by chymotrypsin (1 :200 - 1 :20 w/w) in 50 mM HEPES, pH 7.0, 10 mM CaCl 2 and 100 mM NaCl at 1-40°C for periods of time ranging from 0 to 24 hours.
- Thrombin is a serine protease enzyme (37,000 Da), which selectively cleaves Arg-Gly bonds in fibrinogen. It is involved in the blood coagulation cascade, cleaving fibrinogen to fibrin which forms the blood clot.
- PLC protein kinase C
- Thrombin also plays an important role in promoting mitosis in fibroblasts, chemotaxis in monocytes and neurite retraction in neurons.
- polypeptides are used for the assay of thrombin activity according to the invention:
- Immobilised substrate polypeptide HHHHHHGGIAQLEQEIAQLEQENRQLEQEIAQLEQEI
- R represent the cleavage site for thrombin with the cut occurring between R and Q residues, the 6 His residues are used for immobilisation, and K represents the fluorophore attachment site.
- Binding partner polypeptide IAQLKQKIAQLKQKNAQLKQKIAQLKQKICQLKQK C represents the label attachment site.
- Peptides (0.001-100 ⁇ M) are cleaved by thrombin (1 : 100 - 1 :10 w/w) in 50mM Tris-HCl, pH 7.0, lOOmM NaCl, 2.5mM CaCl 2 , 0.1% 2-mercaptoethanol at 1-40°C for periods of time ranging from 0 to 24 hours.
- Tobacco Etch Virus (TEV) protease- the enzyme is a 27,000Da protein which plays an important role in processing the precursor of viral polyprotein (Mutational analysis of Tobacco Etch Virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase, Carrington, J.C, Cary, S.M. & Dougherty, W.G. (1988) J. Virol, 62 2313-2320). This enzyme is commonly used in processing and cleavage of specific tags which are inserted specifically to aid purification of targeted recombinant proteins.
- TEV protease is assayed in this invention using the following peptides:
- Immobilised substrate peptide HHHHHHGGIAQLEQEIAQLEQENAYLQSEIAQLEQEI AKLEQE
- Residues in bold represent the consensus sequence for TEV site with the cleavage occurring between Q and S residues.
- K represents the fluorophore modification site.
- the 6 His residues are used for immobilisation.
- Binding partner polypeptide IAQLKQKIAQLKQKNAQLKQKIAQLKQKICQLKQK C represents the label attachment site.
- Peptides (0.001-100 ⁇ M) are cleaved by TEV (1 :300 - 1 :30 w/w) in 50mM Tris-HCl (pH 7), 0.5 mM EDTA, 1 mM DTT or 42.2 mM K 2 HPO 4 , 7.8 mM KH 2 PO 4 , 0.5 mM EDTA pH 7.0 at 1-40°C for periods of time ranging from 0 to 24 hours.
- Aminopeptidase M - Peptides (0.001-100 ⁇ M) are cleaved by Aminopeptidase M (1 :50 - 1 :500 w/w) in 42.2 mM K 2 HPO 4 , 7.8 mM KH 2 PO 4 pH 7.0 at 1-40°C for periods of time ranging from 0 to 24 hours.
- Carboxypeptidase Y- Peptides (0.001-100 ⁇ M) are cleaved by Carboxypeptidase Y (1 :100 - 1 :10 w/w) in 50mM sodium citrate pH 6.0 or 42.2mM K 2 HPO 4 , 7.8mM KH 2 PO 4 , 150 mM NaCl at 1-40°C for periods of time ranging from 0 to 24 hours.
- Fluorophore labelled peptides (in a 1 :6 molar ratio of fluorescein-labelled (pepF) to tetramethylrhodamine-labelled (pepR) peptide) are mixed. Samples are analysed in a fluorimeter using excitation wavelengths relevant to pepF ( ⁇ 450nm) and emission wavelengths relevant to pepF ( ⁇ 516nm) and pepR ( ⁇ 580nm). A ratio of emission from pepR over that from pepF following excitation at a single wavelength is used to determine the efficiency of FRET between fluorophores, and hence their spatial proximity. Typically the measurements are performed at 0-37°C as a function of time following the addition of the relevant protease in appropriate buffer as detailed above.
- immobilisation of template peptides on HisSorb and BIAcore supports is based on the specifically engineered 6 histidine residues tail in the peptide or protein of interest, this tail binds specifically to the Ni-Nitrilotriacetic acid ligand bound to an appropriate support.
- a selection of cell cultures such as BHK fibroblasts, Swiss 3T3, or HeLa cells can be utilised to provide lysates.
- Cells are grown according to standard methods and then lysed by lytic agents such as digitonin or saponin. The cells are then centrifiiged and the lysates are resuspended in appropriate buffers which are suitable for measurements of enzymatic activities of interest.
- Cell lysates are added to the microplate wells containing the immobilised peptide template which has the modification site(s), incubated for periods ranging from 1 second to 24 hours. The plates are then washed with buffer and the measure of activity of cellular enzymes is judged by addition of the common partner to the wells (The FRET measurements indicate the presence or lack of modification).
- the assay of the invention is used to detect modification of a polypeptide by chemical phosphorylation.
- 'Zip 3' is a polypeptide which has a phosphorylation site in the centre of the molecule, and has the following amino acid sequence:
- This sequence is derived from amino acids 249-281 of GCN4 (Genbank Accession No.
- the sequence AAK has been added to the C-terminus of that polypeptide sequence, N 6 has been changed to T and R 273 has been changed to C.
- the threonine residue (shown here in bold type) is the residue which is to be phosphorylated.
- the cysteine residue (also shown in bold type) provides the site for attachment of thiol- directed fluorescent labels.
- the circular dichroism (measured in units of ellipticity) of proteins at 222 nm provides a measure of the amount of ⁇ -helix present in the structure, with a large, negative ellipticity indicting a high level of helicity.
- the coiled-coil has a distinctive ⁇ -helical CD spectrum with minima at 222 nm and 208 nm (O'Shea et al., 1989, Science, 243: 538-542).
- the CD spectra of the unmodified Zip3 and its phosphorylated form are determined at a sample concentration of 10 ⁇ M in 150 mM KC1, 42.2 mM K 2 HPO 4 , 7.8 mM KH PO 4 , pH 7.0 at 20°C; spectra are recorded using a 1mm pathlength cell in a Jasco J- 715 spectropolarimeter with a Jasco PTC-348W Peltier temperature control unit.
- Peptide concentration is determined by tyrosine absorbance at 280 nm in 6M GuHCl.
- Peptides are labelled using a method adapted from one known in the art (Hermanson, 1996, Bioconiugate Techniques, Academic Press). 20 mM fluorescein iodoacetamide (FAM) in DMSO and 0.23 mM peptide in 20 mM TES buffer, pH 7.0 are prepared. These are mixed in a molar ratio of (0.9:1.0, label :peptide) and incubated at 4°C in the dark for a minimum of 2 hours.
- FAM fluorescein iodoacetamide
- This method is also applied to labelling of a peptide with rhodamine maleimide at a ratio of (0.9:2.0, label :peptide); This method is also applied to labelling of a peptide using rhodamine iodoacetamide at a ratio of (0.9:1.0, label :peptide). Labelling is assessed by reverse phase HPLC (C18 column; solvent A: H 2 O/0.1% TFA; solvent B: acetonitrile/0.1% TFA) and MALDI-TOF mass spectrometry. Zip3 peptides labelled with fluorescein (Zip3F) and rhodamine (Zip3R) are thus generated.
- FRET occurs between fluorophores attached to unphosphorylated peptides which interact with each other, but that FRET does not occur between fluorophores attached to phosphorylated peptides which do not interact with each other (as evidenced by loss of structure in CD experiments).
- Fluorescence at 516 nm is measured for labelled Zip3 polypeptides in a 1cm pathlength cell at peptide concentrations of 0.08 ⁇ M Zip3F and 0.48 ⁇ M Zip3R in 50 mM KCl, 42.2 mM K 2 HPO 4 , 7.8 mM KH 2 PO 4 , pH 7.0 at 37°C in a Photon Technology Instruments
- Example 1 the utility of interacting modifiable polypeptides as reporters of protein phosphorylation using FRET is demonstrated.
- the application of such polypeptides to the assay of the activity of a polypeptide modification enzyme is illustrated.
- a timecourse of phosphorylation of these peptides by PKA is run in 50 mM histidine HCl (pH 7.0), 5 mM MgSO 4 , 5 mM NaF, 0.05 mM EGTA, 120 mM KCl with 0.2 mM ATP (30.9 cpm/pmol 32 P-ATP) and 0.5 ⁇ M PKA.
- the results show that Zip 4S is a good substrate for the PKA enzyme, with a rate of phosphorylation comparable to that seen with a known substrate (PL919Y, a Phospholamban peptide; Drago and Colyer, 1994, J. Biol. Chem.: 269: 25073-25077).
- Zip4T is also phosphorylated, at a slower rate, with full phosphorylation achieved in 30 minutes in this assay.
- the difference in cpm recovered between the Zip4 peptides and PL919Y is due to the difference in recovery of these peptides on P81 paper.
- the plateau indicates full phosphorylation.
- a set of timecourses of phosphorylation is performed on Zip4S using PKA and Ca 2+ /Calmodulin-dependent Protein Kinase (CaMK-II), together with positive and negative controls for CaMK-II (these controls confirm that the preparation of CaMK-II possesses the expected characteristics of that enzyme).
- PKA phosphorylation is performed in 50 mM histidine/HCl (pH 7.0), 5 mM MgSO 4 , 5 mM NaF, 0.05 mM EGTA, 120 mM KCl with 0.2 mM ATP (39.72 cpm/pmol 32 P-ATP) and 0.25 ⁇ M PKA.
- CaMK-II phosphorylation is performed in 50 mM histidine/HCl (pH 7.0), 5 mM MgSO 4 , 5 mM NaF, 120 mM KCl, 0.5 mM Ca 2+ , 0.037 mg/ml calmodulin with 0.2 mM ATP (39.72 cpm/pmol 32 P-ATP) and 10% crude CaMK-II.
- Ca 2+ -free experiment Ca 2+ is replaced by 0.05 mM EGTA.
- the results indicate that the phosphorylation site in Zip4S is recognised only by C-PKA. CaMK-II is unable to phosphorylate Zip4S, while PKA mediates complete phosphorylation of that protein.
- Such specificity of a modification site in a polypeptide according to the invention is a clear advantage for use of the invention in an intracellular assay, or for measuring the activity of a particular polypeptide modifying agent in a complex mixture such as a cell lysate.
- Fluorescence is used to report on the phosphorylation status of these peptides.
- a loss of emission at 516 nm is seen on addition of Zip4SR to Zip4SF, as FRET occurs when the differentially-labelled polypeptide partners are allowed to associate. Emission at around 575 nm also increases; whilst part of this increase may be attributable to direct excitation of the rhodamine label, the increase is consistently above that seen in the phosphorylated scan, suggesting that at least a proportion of the increase is due to excitation by FRET.
- emission at 516 nm returned to above the level produced by Zip4SF alone, while emission at 573 nm decreases slightly; this decrease accounts for the amount of FRET-derived emission which is lost. No loss of FRET is observed on addition of PKA in the absence of ATP.
- Pep 1 is a polypeptide which has a phosphorylation site in the centre of the molecule and has the following amino acid sequence:
- This sequence is based on a wholly synthetic coiled coil structure according to O'Shea. E. K. et al, (1993). Current Biology 3, 658-667 and Nautiyal. S. at al, (1995) Biochemistry 34, 11645-11651.
- the sequence has poly-histidine insert added to its N terminus for immobilisation pu ⁇ oses and the two glycine residues are added as a spacer.
- the amino acid sequence in bold is inserted as a specific phosphorylation recognition site and the underlined lysine residue is inserted for labelling pu ⁇ oses.
- Pep 2 is a polypeptide which has no kinase modification sites and is synthesised to form a heterodimeric partner with Pep 1.
- the underlined cysteine residue is inserted for a label attachment pu ⁇ oses.
- Pep 1 associates with its partner Pep 2 to form a coiled coil oligomer.
- Ni-NTA derivatised sensor chip (the chip is commercially available from BIAcore, Uppsala, Sweden; the NTA ligand is available from Quiagen GmbH) is first washed with eluent buffer containing lOmM HEPES, 150mM NaCl, 50 ⁇ M EDTA, o.oo5% Surfactant
- the partner peptide Pep 2 (0.01-100 ⁇ M in HEPES buffer) is then applied to the sensor chip. At this stage the sensorgram is recorded for the change in mass. • When Pep 1 is unphosphorylated, Pep 2 associates with Pep 1, Thus giving an increase in mass which is observed via a positive signal of the sensorgram. This is indicative of the absence of PKA.
- the assay is to be performed using an alternative output method, such as FRET, a number of general techniques and test procedures are used. These are set out below, and find use in other embodiments of assays of this type.
- Peptides are labelled using a method adapted from one known in the art (Hermanson, 1996, Bioconjugate techniques. Academic Press). 20 mM fluorescein 5-EX succinimidyl ester in 50 mM sodium carbonate pH 8.5 and 0.23 mM peptide in 20 mM TES buffer, pH 7.0 are prepared. These are mixed in a molar ratio of 0.9:1.0 (labehpeptide) and incubated at 4°C in the dark for a minimum of 2 hours.
- the method is also applied to labelling of a peptide with rhodamine maleimide and rhodamine iodoacetamide at a ratio of 0.9: 1.0 (label :peptide). Labelling is assessed by reverse phase HPLC (C18 column; solvent A: H 2 O/0.1% TFA; solvent B: acetonitrile/0.1% TFA) and MALDI-TOF mass spectrometry. Pep 1 peptide labelled with fluorescein (Pep IF) and Pep 2 peptide labelled with rhodamine (Pep 2R) are thus generated.
- Immobilisation of Pep IF peptide on the Ni-NTA derivatised microtitre plate is based on the specifically engineered 6-histidine residue tag in Pep 1. This tail binds specifically to the Ni 2+ ions attached to NTA. The Nitrillotriacetic acid ligand is bound to the plate surface according to manufacturer's instructions. Initially the plates are washed with PBS buffer containing 50 ⁇ M EDTA (enough to remove contaminating metal ions without stripping the Ni 2+ ) and then the plates are charged with Ni 2+ (lOO ⁇ M in PBS buffer).
- Pep IF (0.01-100 ⁇ M in PBS buffer) is added to the microplate wells (the capacity of binding is determined by the degree of derivatisation of Ni-NTA) and incubated for 1 to 2 hours at room temperature or overnight at 4°C The unbound peptide is then removed by aspiration followed by a thorough wash with excess buffer. Excess wash buffer is removed by gentle tapping of the inverted plates on paper towels.
- Pep 1 and Pep 2 labelled as described above are assayed for association or dissociation upon addition of the modifying agent by FRET as follows:
- Pep IF peptide labelled with fluorescein
- a fluorimeter cuvette containing 2ml of 50mM potassium phosphate pH 7.2 in the presence of 120mM KCl, 5mM NaF, 5mM MgSO 4 , 0.05mM EGTA and 2mM ATP, and incubated for 10 minutes to reach thermal equilibrium at 37°C.
- Fluorescence emission of fluorescein is measured at 520nm by exciting at appropriate wavelength (450nm).
- Pep 2R (Pep 2 labelled with rhodamine) at concentrations equal to that of Pep IF (0.5 ⁇ M) is added and the fluorescence emission of Pep IF and Pep 2R are monitored.
- Pep 2 associates with Pep 1 causing the fluorophores to approach each other.
- some of the energy emitted by fluorescein attached to Pep 1 is absorbed by rhodamine attached to pep2 peptide leading to fluorescence decrease.
- the circular dichroism (measured in units of ellipticity) of proteins at 222nm provides a measure of the amount of ⁇ -helix present in the structure, with a large, negative ellipticity indicating a high level of helicity.
- the coiled coil has a distinctive ⁇ -helical CD spectrum with minima at 222nm and 208nm (O'Shea et al. 1989. Science, 243, 538-542).
- the CD spectra of the unmodified peptide Pep 2 and its phosphorylated partner Pep 1 are determined at a sample concentration of lO ⁇ M in 50mM Tris.HCl, pH 7.2 and 120mM KCl at 37°C
- Pep IF is immobilised on a Ni-NTA microplate, as described above.
- Pep2R is immobilised on a Ni-NTA microplate, as described above.
- Pep IF is immobilised on a Ni-NTA microplate, as described above. Phosphorylation of Pep IF is initiated by the addition of 1.0 ⁇ M PKA, incubated for different time periods (Osec- lhour) in different wells containing the assay at room temperature in a buffer containing 50mM potassium phosphate buffer pH 7.2; 150mM NaCl, 5mM NaF, 5mM MgSO 4 and 0.5mM ATP. Phosphorylation is then stopped by the addition of 5 ⁇ M PKI (5-22)-amide, a specific PKA inhibitor (J. Biol. Chem. (1989) 264:8802-8810) and the solution reagents by aspiration.
- Pep 2R (0.01-100 ⁇ M, with different amounts in different wells) is added to each well and incubated for 5min at room temperature. Excess Pep 2R solution is removed by aspiration and FRET is measured by excitation at 450nm and emission at 580 and 520nm. The ratio of 580/520 is used to record FRET. The time course of phosphorylation can be reconstructed from the FRET measurements in separate wells of the plate which are exposed to the kinase activity for different periods of time.
- CaMK-II is an enzyme which consists of subunits of 50,000, 58,000-60,000 Da in a 3: 1 ratio. It is a multifunctional enzyme which is dependent on Ca 2+ and calmodulin for its activity. It is involved in mediating various cellular processes in response to second messenger signalling stimuli including gene expression, neurotransmitter synthesis and release, calcium homeostasis, ion channel function, carbohydrate metabolism and cytoskeletal function (Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559) The consensus sequence required for recognition for CaMK-II is RXXSX.
- Immobilised substrate polypeptide HHHHHHGGIAQLEQEIAQLRQESAQLEQEIAQLEQEI AKLEQE
- Binding partner polypeptide IAQ KQEIAQLKQKNAQLKQKIAQLKQKICQLKQK C represents the label attachment site.
- the peptides are labelled as in Example 1.
- the substrate peptide is immobilised as in Example 3.
- Phosphorylation of immobilised peptides (0.01-100 ⁇ M) by CaM kinase II is carried out in 50mM Histidine buffer pH 7.0, lOmM MgSO4, 120mM KCl, 5mM Sodium Fluoride, in the presence or absence of Ca 2+ /CaM.
- the reaction is initiated by the addition of 0.2mM ATP at 30-37°C and phosphorylation is measured as described in Example 3.
- S6 Kinase is a single polypeptide chain (57,000Da), playing an important role in protein synthesis. It exerts its function by phosphorylating the S6 P70 ribosomal protein.
- various hormonal stimuli such as insulin (Novak-Hofer, I., & Thomas, G. (1984) J Biol. Chem. 25, 5995-600., Nemenoff, R. A., Gunsaius, J.R., and Avruch, J. (1986) Arch. Biochem. Biophys. 245, 196-203).
- growth factors Novak-Hofer, I., & Thomas, G. (1984) J. Biol. Chem.
- Immobilised substrate polypeptide HHHHHHGGIAQLEQEIARLRQESAQLEQEIAQLEQEI AKLEQE
- Binding partner polypeptide IAQLKQEIAQLKQKNAQLKQKIAQLKQKICQLKQK C represents the label attachment site.
- the peptides are labelled as in Example 1.
- the substrate peptide is immobilised as in Example 3.
- Immobilised peptides (0.01-100 ⁇ M) are incubated in a buffer containing 20 mM MOPS pH 7.2, 25 mM ⁇ -glycerol phosphate, ImM Sodium orthovanadate and 1 mM dithiothrietol, in the presence of a cocktail of kinase inhibitors for PKA, PKC and CaM kinase known in the art.
- the reaction is initiated by the addition of 500 ⁇ M Mg-ATP and phosphorylation is measured as described in Example 3.
- the peptide sequence shown below is based upon the p67 ⁇ glycosylation acceptor site S-316 (Reason et al., 1992, supra) and the adaptor of that sequence, which have been modified to improve their compliance with a coiled-coil sequence pattern:
- SAVSSADGTVL Zip5 HHHHHHGGIAQLEQEIAQLSAV-SSALGTVIAQ EQEIAKLEQE
- the site of O-glycosylation is S-316 ('a' position of heptad 3), the sequence is based on that of p67 SRF (313-323) sequence except for (D 319 — -> L), and has undergone an extension of its sequence with sequence which complies with the canonical coiled-coil sequence to complete five heptads, and has an N-terminal 6-His residue extension in order to immobilise the peptide as in Example 3.
- Binding polypeptide IAQLKQKIAQLKQKNAQLKQKIAQLKQKICQLKQK Fluorophores are attached to the single cysteine residue (shown in bold) or the single lysine residue in each polypeptide as in Example 3, the Zip5 polypeptide is immobilised and used as in Example 3.
- polypeptides form stable oligomers in the absence of a glycosylated serine as assessed by FRET.
- O-glycosylation of Zip5 occurs upon exposure of this peptide to a source of a protein- modifying enzyme which mediates O-Glycosylation of peptides (e.g. uridine diphospho- N-acetylglucosamine:peptide ⁇ N-acetylglucosaminyltransferase) under the appropriate conditions (defined in Haltiwanger et al., 1990, J. Biol. Chem., 265:2563-2568) causes modification of the Ser residue shown in bold and, consequently, the dissociation of these polypeptides and loss of FRET.
- a protein- modifying enzyme which mediates O-Glycosylation of peptides (e.g. uridine diphospho- N-acetylglucosamine:peptide ⁇ N-acetylglucosaminyltransferase) under the appropriate conditions (defined in Haltiwanger et al., 1990, J. Biol. Chem., 265:
- Chymotrypsin is a digestive enzyme found in the small intestine.
- Peptides based on a heterodimeric coiled coil provide a reporter molecule able to monitor cleavage by chymotrypsin.
- Peptide 1 is synthesised by automated peptide synthesis.
- cysteine residue shown in bold, provides a site for attachment of a thiol-directed fluorescent label.
- This peptide is capable of binding to the immobilisable partner polypeptide, peptide A:
- the tyrosine residue (shown here in bold type) is the residue at which chymotrypsin cleaves.
- the lysine residue (also shown in bold type) provides the site for attachment of thiol-directed fluorescent labels.
- the 6-His residues are used for immobilisation of the polypeptide as in Example 3.
- lyophilised chymotrypsin is dissolved in ImM HCI.
- Polypeptides immobilised as in Example 3 are cleaved by chymotrypsin (1 :50 w/w) in 50 mM HEPES, pH 7.0, 10 mM CaCl 2 , 100 mM NaCl at 37°C for 2 hours.
- the reaction is monitored as described in Example 3.
- Addition of chymotrypsin to peptides AF and AR eliminates FRET, as a result of disruption of coiled coil formation.
- Exopeptidases are important in the breakdown of peptides after initial digestion by endopeptidases such as chymotrypsin or trypsin.
- the end product of attack by such enzymes are free amino acids or dipeptides.
- An assay for this type of proteolysis relies upon the fluorophores being far from the initial site of proteolysis such that digestion ultimately results in a fragment which is too small to remain within the coiled coil multimer, at which point FRET is lost.
- An unblocked N-terminus is essential for activity of aminopeptidases.
- assays for amino- and carboxypeptidase digestion are set up comprising as the reporter peptides N and C:
- peptide C the partner peptide D is used:
- peptide N the partner peptide E is used: LKQKNAQLKQKIAQLKQKICQLKQK Peptide E
- Peptides C, N, D and E are labelled as set forth in Example 3, at the C and K residues as before.
- Substrate peptides are then immobilised on physical supports as described in Example 3.
- fluorescein-labelled peptides N and C are assayed for FRET in the presence of rhodamine-labelled peptides D and E. In both cases, FRET is observed. Addition of carboxypeptidase abolishes FRET for the peptide C-D pair; however, FRET for the peptide N-E pair is not affected.
- This protease is important in the processing of the precursor polyprotein of TEV (Mutational analysis of Tobacco Etch Virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase, Carrington, J.C, Cary, S.M. & Dougherty, W.G., Journal of Virology (1988) 62 2313-2320).
- the peptides used are:
- Peptide 2 is constructed to contain the TEV protease cleavage site, and labelled at an inserted unique lysine residue. Immobilisation is carried out as in Example 3.
- the proteolytic reactions are performed as follows:
- Peptide 1 is immobilised, as described, and cleaved with TEV (1 :100 w/w) in 50mM Tris.HCl (pH 7.0) at 37°C for 2 hours.
- the TEV activity is assessed by monitoring FRET as in Example 3.
- Thrombin is the final enzyme in the blood clotting cascade, cleaving fibrinogen to fibrin. Its effects are, in part, regulated by a negative feedback mechanism in which thrombin activates protein C which ultimately switches off the cascade and therefore thrombin production. This enzyme is also important in promoting mitosis in fibroblasts, chemotaxis in monocytes and neurite retraction in neurons.
- a heterodimeric coiled coil is provided to monitor cleavage by thrombin in the manner described above. In order to have cleavage at one site only it is necessary to eliminate all arginine residues other than that at the intended cleavage site from the peptide (as in Peptide 3 below).
- Thrombin cuts between the bold arginine residue (R) and glutamine (Q).
- the peptide is labelled at the bold lysine residue.
- the partner peptide used is peptide 33, labelled at the cysteine shown in bold:
- IAQLKQKIAQLKQKNAQLKQKIAQLKQKICQLKQK peptide 1 Peptides are immobilised as described in Example 3 and cleaved with thrombin (1 :50 w/w) in 50mM Tris.HCl, pH 7.0, lOOmM NaCl, 2.5mM CaCl 2 , 0.1% ⁇ -mecaptoethanol, at 37°C for 2 hours.
- the activity of the thrombin is monitored by assessing FRET as in Example 3.
- FRET is observed between peptides 3 and 1 , labelled with fluorescein and rhodamine respectively, in the absence of thrombin. Incubation with thrombin as above abolishes FRET, due to disruption of the coiled coil and loss of association between the polypeptides.
- the caspases are a family of proteases with an absolute requirement for cleavage after aspartic acid. They are synthesised as inactive proenzymes and cleaved either autocatalytically or by another member of the family to form an active heterodimer of a large and a small subunit. This family of enzymes play a role in inflammation but, more importantly, several members of the family provide signals triggering apoptosis of the cell and others are directly involved in cell disassembly itself.
- the role of caspases in the apoptotic process includes the inactivation of proteins protective against apoptosis such as the Bcl-2 proteins, the destruction of proteins with a key role in cell structure such as the lamins and the deregulation of proteins by the disruption of links between regulatory and effector domains.
- a loss of control of apoptosis leading either to excessive or inadequate cell death plays a role in many disease processes including cancers, neurodegenerative disorders and auto-immune disease. Evidence is already available suggesting that caspase inhibitors are able to protect against inappropriate cell death.
- a heterodimeric coiled coil reporter molecule is provided to monitor cleavage by members of the caspase family (e.g. for Caspase 2, 3, 7 below) in the manner described above.
- the immobilised peptide has the following structure: HHHHHHGGIAQLEQEIAQLEDENDQLEQEIAQLEQEIAKLEQE Peptide 4
- binding partner is:
- Example 10 The procedure of Example 10 is repeated with Peptides 4 and 1 , using caspase 2 in place of thrombin, and identical results are obtained.
- the assay format according to the invention can be used to measure post-translational modification events which have proteolysis as an integral step.
- the molecular basis of these assays is the destabilisation of the binding partnership following the proteolytic event which, if coincident with the covalent addition of another group (farnesylic acid etc.), provides an indirect reporter function for these classes of posttranslational modification.
- the post-translational modification of proteins with fatty acids includes the attachment of myristic acid to the primary amino group of an N-terminal glycine residue (Milligan, G.,
- Fatty acylation of proteins is a dynamic post-translational modification which is critical for the biological activity of many proteins, as well as their interactions with other -84- proteins and with membranes.
- the location of the protein within a cell can be controlled by its state of prenylation (fatty acid modification) as can its ability to interact with effector enzymes (Ras and MAP kinase, Itoh, T., Kaibuchi, T., Masuda, T., Yamamoto, T., Matsuura, Y., Maeda, A., Shimizu, K., & Takai, Y. (1993) J. Biol. Chem.
- Ras and adenylate cyclase in yeast
- the prenylation status of Ras is important for its oncogenic properties (Cox, 1995, above) thus interference with the prenylation status of Ras is considered a valuable anti-cancer strategy (Hancock, J.F. (1993) Current Biology 3, 770)
- An engineered heterodimeric coiled coil is designed to produce a homodimeric reporter molecule capable of monitoring geranylgeranylation.
- the features are (i) a short coiled- coil structure to achieve a coiled coil oligomer of reasonable stability; (ii) modified C- terminal residues as necessary to comply with the substrate recognition features of the enzyme under study, geranylgeranyl transferase (GGTase I).
- Geranlygeranylation is then performed by incubating immobilised peptide 5 with 25 ⁇ l rabbit reticulocyte lysate (or the equivalent quantity of either a purified geranylgeranyltranferase or a test sample) and 40 ⁇ M geranylgeranyl pyrophosphate in a final volume of at least 50 ⁇ l (Cox 1995, above). Peptide 5a is then added at a concentration or 50 ⁇ M and the modification of the reporter is followed by observation of acceptor/donor fluorescence emission upon excitation of the donor fluorophore at an appropriate wavelength. A decrease in this ratio indicates a loss of FRET which is a measure of the geranylgeranylation of the reporter.
- the assays of the invention In order to configure the assays of the invention not only to monitor chemical modification in defined systems, but also to screen for activities in complex environments (e.g. whole cell lysates) it may be necessary to protect the reporter molecule from proteolysis at sites other than those engineered into the structure.
- the vast majority of naturally occurring enzymes will not recognise as substrates polypeptides comprising D amino acids.
- the specificity of the reporter group is increased by limiting the use of L amino acids (recognised by enzymes) to residues involved in recognition by the protease. This greatly reduces the probability of unplanned proteolysis at additional sites by other proteases in, for example, a cell extract.
- Proteins or peptides composed of D amino acids have a structure which is the exact mirror image of that formed with L amino acids (Identification of D-peptide ligands through mirror image phage display, Schumacher, T.N.M., Mayr, L.M., Minor, D.L. Jnr., Milhollen, M.A., Burgess, M.W., Kim, P.S., Science (1996) 271 1854-1857). We therefore conclude that a coiled coil reporter designed in this way will have a structure of the form shown in Figure 20.
- Example 14 Biotin/streptavidin immobilisation of CaMK reporter peptides
- the binding of a peptide to a streptavidin membrane and subsequent modification-dependent binding of a partner polypeptide is demonstrated.
- the assay is configured as a homodimer assay, but only one partner is modified.
- the calmodulin-dependent protein kinase II (CaMK-II) peptide used in this assay is:
- Biotin-BMCC (l-Biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane) is prepared at 3 mg/ml in DMSO. Both the dephosphorylated and chemically phosphorylated CaMK-II reporter peptides are prepared at 1 mg/ml in 20mM TES buffer pH 7.0. The biotin is diluted 1 :25 (v/v) into each peptide solution (this gives a final ratio of 1 :1, peptide:biotin). Labelling is allowed to proceed overnight at room temperature. The success of the labelling reaction is confirmed by MALDI-TOF mass spectrophotometric analysis.
- a lane of biotin labelled dephosphorylated (unmodified) and a lane of biotin labelled phosphorylated (modified) peptide is spotted on to a streptavidin membrane (lO ⁇ g, 3 ⁇ g, 1 ⁇ g, 0.3 ⁇ g, 0.1 ⁇ g, 0.03 ⁇ g, 0.01 ⁇ g in each lane).
- the membrane is washed for 2x5 minutes in 2%BS A/PBS (w/v) and blocked in this solution for 1 hour.
- the membrane is then incubated with 30 ⁇ g rhodamine labelled CaMK-II dephosphorylated peptide (prepared as described in example 3) in 2ml 2%BSA/PBS for 1 hour at room temperature with rocking.
- the membrane is washed for 3x5 minutes in 2%BSA/PBS (w/v).
- the dephosphorylated CaMK-II peptide probe hybridises with the dephosphorylated peptide at amounts from lO ⁇ g peptide applied in 10 ⁇ l to 0.1 ⁇ g applied in 1 ⁇ l. The phosphorylated peptide is not detected by the probe. These data show that the dephosphorylated probe can detect immobilised dephosphorylated peptide but not phosphorylated peptide in a membrane format.
- Example 15 Measurement of protein kinase and phosphatase activities using an immobilised assay with binding partners labelled with GFP or chemical fluorophores.
- Src protein kinase activity may be determined by measuring the transfer of phosphate groups to immobilised substrates, which may be natural binding domains or peptide substrates based on natural binding domains. The phosphorylation of the immobilised substrate is then detected by the binding of a partner peptide or protein domain, labelled with a fluorophore. The binding of the partner is phosphorylation dependent i.e. binding is either increased or decreased depending on the phosphorylation state of the immobilised substrate.
- the assay of kinase or phosphatase activity can be adapted to an endpoint assay format where excess incoming binding partner can be washed away.
- the assay requires that only the non-immobilised partner be labelled with a fluorophore, the other should be tagged with a suitable anchoring moiety (e.g. for immobilisation through a biotimavidin interaction or a His-Tag:Ni NTA interaction).
- the assay can be used to determine inhibitors of the binding interaction or of the phosphatase/kinase involved in mediating the interaction.
- Example 16 Assay of Src kinase using an immobilised assay with natural binding partners labelled with a fluorescent protein.
- Src kinase activity can be detected by the phosphorylation of a peptide derived from the T-cell receptor (TCR) ⁇ chain.
- TCR T-cell receptor
- the peptide is immobilised via a biotin/streptavidin interaction, and phosphorylation is detected by the binding of a GFP labelled SH2 domain from the ZAP70 kinase (ZAP-GFP).
- TCR peptide sequences Peptide 1.
- Phosphorylated TCR ⁇ chain Peptide 1.
- TCR peptide immobilisation methods Peptide 1 or peptide 2 are biotinylated under mild conditions using amine or thiol directed chemistry (20mM TES pH 7 for thiol directed labelling, and 200mM sodium bicarbonate pH 8.3 for amine directed labelling) using 230 ⁇ M peptide in the presence of 200 ⁇ M biotin.
- Peptides are immobilised using a 96-well streptavidin coated plate ABgene using l.O ⁇ g/ml peptide in 200 ⁇ l TBS containing 0.05% Tween 20 (TBST) and 1% BSA TBST/BSA. Excess peptide is washed off with 3 washes of 200 ⁇ l of TBST.
- the BFP-TCR protein is expressed and purified with a hexapeptide comprised of histidine residues to facilitate anchoring the protein to a microtitre plate coated with a nickel chelate surface (Pierce). Immobilisation of the domain is achieved simply by incubating the domain on the plate in binding buffer( 50mM phosphate pH7, 300mM NaCl, 5mM imidazole). Excess protein is washed off with the binding buffer including 0.2%o (v/v) Tween 20 using 3 washes of 200 ⁇ l.
- ZAP-GFP Primers are designed based on the published ZAP-70 DNA sequence (Genbank accession number L05148). The SH2 domain (amino acids 1-259) of ZAP70 is cloned by PCR using the following oligo-nucleotides: Forward primer GGGATCCATATGCCAGACCCCGCGGCGCACCTG Sequence 1 Reverse Primer GGAATTCGGGCACTGCTGTTGGGGCAGGCCTCC Sequence 2
- the resultant PCR fragment is digested with BamHl and EcoRl and inserted into pET28a (Novagen) to generate vector pF545.
- DNA encoding GFP in the vector pQBI25-FNI (Quantum) is digested with Mlul and the resultant 5' overhang is "filled in” using T4 DNA polymerase (NEB).
- T4 DNA polymerase (NEB)
- the DNA is further digested with EcoRl and the resultant 850 bp band is gel purified.
- the vector pF545 is digested with Hindlll and the resultant 5' overhang is "filled in” with T4 DNA polymerase and then further digested with EcoRl.
- the digested vector is gel purified it is ligated with the purified DNA encoding GFP to generate pFS46 which is designed to express a ZAP70-GFP fusion protein in bacteria.
- BFP-TCR ⁇ Primers are designed based on the published T-cell receptor Zeta chain DNA sequence (Genbank accession number J04132). Three different lengths of the TCR are cloned by PCR (corresponding to residues 52-163, 60-163 & 69-163) using the following oligo-nucleotides:
- the resultant PCR fragment is digested with Apal and BamHl and inserted into pQBI50- fNl (Quantum) downstream of DNA encoding BFP.
- DNA encoding the BFP-TCR ⁇ fusion protein is isolated on a Nbel BamHl fragment and ligated into p ⁇ T28a digested with the same enzymes. This generates bacterial expression vectors for the BFP- TCR ⁇ fusion proteins (pFS48 TCR 52-163; pFS49 TCR 60-163; pFS50 TCR 69-163).
- Fresh transformants of ZAP-GFP pET-28a in BRL(DE3) and BFP-TCR pET-28a in BRL(DE3) pLysS are used to inoculate 3ml LB/kanamycin (lOO ⁇ g/ml).
- the starter cultures are incubated overnight at 37°C with shaking. From these starter cultures lml is used to inoculate 400ml Terrific Broth kanamycin (lOO ⁇ g/ml) in a 2L, baffled flask. Cultures are incubated at 37°C at 200 ⁇ m for approximately 5hrs until the OD600nm have reached 0.5Abs units. At this point cultures are induced by adding IPTG to a concentration of ImM. The cultures are then left incubating at room temperature overnight with gentle shaking on a benchtop rotator.
- Bacteria are harvested by centrifugation at 3000 rpm for 20min.
- the bacterial pellet is resuspended in 25ml lysis buffer (50mM Pi pH 7.0, 300mM NaCl, 2% Proteinase inhibitor cocktail (Sigma), 0.75mg/ml Lysozyme). Lysis of the resuspended cells is initiated by gentle stirring for lhr at room temperature.
- the partially lysed mixture is subjected to 2 cycles of freeze thawing in liquid nitrogen.
- the cells are sonicated on ice using a 10mm probe at high power. Sonication is performed on a pulse setting for a period of 3 min.
- the crude lysate is then centrifiiged at 15000 ⁇ m for 30min to remove cell debris.
- His tagged proteins are purified from the clear lysate using TALON® resin (Clontech). Proteins are bound to the resin in a batchwise manner by gentle shaking at room temperature for 30 min. Non-His tagged proteins are removed by washing the resin at least twice with lOx bed volume of wash buffer (50mM sodium phosphate pH 7.0, 300mM NaCl, 5mM fluorescence-blank Imidazole).
- the washed resin is loaded into a 2 ml column and the bound proteins released with elution buffer (50mM sodium phosphate pH 7.0, 300mM NaCl, 150mM florescence-blank Imidazole). Elution is normally achieved after the first 0.5ml and within 2-3ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10% glycerol.
- elution buffer 50mM sodium phosphate pH 7.0, 300mM NaCl, 150mM florescence-blank Imidazole. Elution is normally achieved after the first 0.5ml and within 2-3ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10% glycerol.
- FIG. 1 The extent of phosphopeptide binding and detection by ZAP-GFP is shown in Figure 1.
- Peptide 1 is immobilised as described above, and the amount varied from 0-1380 pmoles per well. Immobilised peptide is detected using 200 ⁇ l of ZAP-GFP at l ⁇ M in TBST/BSA and incubating for 1 hour at RT with gentle rocking. This is followed by 6 washes of 200 ⁇ l TBST.
- Bound ZAPGFP is detected by reading the fluorescence of GFP using excitation at 485nm and measuring emission at 520nm. This figure shows that maximal binding and detection is achieved using 46-138 pmoles of peptide, equivalent to 200 ⁇ l of 1-3 ⁇ g/ml peptide.
- Src enzyme is from Upstate Biotechnology. Src kinase, 1 unit, is added to the immobilised peptide 2 substrate, in a 50 ⁇ l total volume of Src kinase buffer (20 mM Tris-HCl pH7.2, 0.5 mM ATP, 10 mM MgCl 2 , 0.006% (v/v) Brij 35, 0.02 mg/ml BSA, 0.04%) (v/v) ⁇ -mercaptoethanol) and incubated for up tol hour at 37°C. The enzyme is washed off with 3 washes of 200 ⁇ l TBST/BSA.
- Phosphorylation is detected by adding 200 ⁇ l of ZAP-GFP at l ⁇ M in TBST/BSA and incubating for 1 hour at RT with gentle rocking. This is followed by 6 washes of 200 ⁇ l TBST.
- Bound ZAPGFP is detected by reading the fluorescence of GFP using excitation at 485nm and measuring emission at 520 nm.
- Wells containing no immobilised pepetide (buffer only) or TCR peptide synthesized with phosphotyrosine in place of tyrosine (peptide 1) are used as a controls ( Figure 2.)
- the immobilised assay is also used to determine the IC 5 o of staurosporine, a general protein kinase inhibitor.
- the kinase assay is performed as above, using 0.25 Units of Src and incubating the enzyme with the immobilised peptide 2 for 30 minutes at 37°C. The results are shown in Figure 5.
- Yersinia phosphatase can be detected by the dephosphorylation of a peptide derived from the T-cell receptor (TCR) ⁇ chain.
- TCR T-cell receptor
- the peptide is immobilised via a biotin/streptavidin interaction, and dephosphorylation is detected by the decrease in binding of a GFP labelled SH2 domain from the ZAP70 kinase.
- TCR peptide sequences See example 16.
- Yersinia protein tyrosine phosphatase is from Upstate Biotechnology. Peptide 1 is immobilised as described above. YOP, 0.003 units, is added to reaction buffer to give a final volume of 50 ⁇ l and final buffer concentrations of 25 mM Tris-HCl pH 7.2, 5 mM EDTA 10 mM ⁇ -mercaptoethanol, 50 ⁇ g/ml BSA. The YOP reactions are incubated at room temperature for 10 minutes. Phosphorylation is detected by adding 200 ⁇ l of ZAPGFP at 1 ⁇ M in TBST/BSA and incubating for 1 hour at RT with gentle rocking. This is followed by 6 washes of 200 ⁇ l TBST. Bound ZAP-GFP is detected by reading the fluorescence of GFP using excitation at 485nm and measuring emission at 520 nm. Dephosphorylated TCR peptide (peptide 2) is used as a control.
- Figure 6 shows a time course of YOP dephosphorylation of peptide 1.
- Figure 7 shows that the assay can be used to measure different amounts of YOP enzyme conditions as above using 0.0.003 units YOP.
- the immobilised assay for YOP is also used to determine the IC 50 of ortho-vanadate, a general protein phosphatase inhibitor.
- the YOP assay is performed as above, using 0.003 units of YOP and incubating the enzyme with the immobilised peptide 1 for 10 minutes at room temperature.
- Sodium vanadate is added to the reaction mix prior to enzyme to give final concentrations of (0.1-30 mM). Results indicate an IC 5 o for vanadate of 0.5 ⁇ M and are shown in Figure 8.
- the kinase domain of Fyn kinase is cloned by RT PCR from mRNA isolated from HeLa cells.
- the Thermoscript RT-PCR kit (Gibco BRL) performs cDNA synthesis and PCR.
- the primers used in both cDNA synthesis and PCR are shown below.
- the resulting PCR product is 840bp corresponding to the size of the kinase.
- the product is digested with BamHVHindlll and ligated into pBluescript SK 11+
- kinase reactions are carried out in 50 ⁇ l volumes.
- Various amounts of Fyn prep (0-20 ⁇ l) are added to reactions consisting of immobilised peptide 2 and a final concentration of buffer components as follows (20 mM Tris-HCl pH7.2, 0.5 mM ATP, 10 mM MgCl 2 , 0.006%) (v/v) Brij 35, 0.02 mg/ml BSA, 0.04% (v/v) ⁇ -mercaptoethanol) in a total volume of 50 ⁇ l in a 96-well plate.
- Fyn is added last to start the reaction, and incubated at 37°C for 1 hour.
- the wells are washed 3 times with 200 ⁇ l of TBST/BSA.
- As a control an assay using 1 unit of Src enzyme is run in parallel in the same conditions. Immobilised peptide 1 is also tested as a control sample.
- Phosphorylated peptide 2 is detected by addition of 200 ⁇ l of ZAP-GFP (approximately 1 ⁇ M) in TBST/BSA and incubation for 1 hour at room temperature. Wells are washed 6 times with 200 ⁇ l TBST. Fluorescence of bound ZAP-GFP is read in 200 ⁇ l TBST, using excitation at 485nm, and measuring emission at 520nm. (See Figure 9).
- Example 19 Assay of Src kinase using an immobilized assay with a natural binding partner (SHP-2) labelled with a fluorescent protein.
- Src kinase activity can be detected by phosphorylation of a peptide derived from SHPS-1.
- the peptide is immobilized via a biotin streptavidin interaction, and phosphorylation is detected by the binding of a GFP labelled tandem SH2 domain from the protein tyrosine phosphatase SHP-2.
- the resultant PCR fragment is digested with Xbal and EcoRl, gel purified and ligated into pET28a (Novagen) to generate vector pFSl 14.
- the validity of the construct is confirmed by sequence analysis.
- DNA encoding GFP in the vector pFS46 is isolated by digestion with the restriction enzymes EcoRI and Xhol and the resultant 860bp band is gel purified and ligated into pFS114 to generate a bacterial expression vector for production of the fusion protein SHP-2-GFP (pFS 115).
- Biotinylated peptides are immobilised in a 96-well streptavidin coated plate (Advanced Biotechnologies) using 1.0 ⁇ g/ml peptide in TBS containing 0.05% Tween 20 and 1% BSA (TBST/BSA).
- TBS containing 0.05% Tween 20 and 1% BSA
- peptide 200 ⁇ l is incubated in the wells for lhr at room temperature with gentle agitation on a rotating platform. Excess peptide is removed with 3 washes of 200 ⁇ l of TBST/BSA.
- Src kinase (Upstate Biotechnology), 1 unit, is added to the immobilised SHPS-1 peptide substrate, in a 200 ⁇ l total volume of Src kinase buffer (20 mM Tris-HCl pH7.2, 0.5 mM ATP, 10 mM MgCl 2 , 0.006% (v/v) Brij 35, 0.02 mg/ml BSA, 0.04% (v/v) ⁇ - mercaptoethanol) and incubated for 1 hour at 37°C The enzyme is removed with 3 washes of 200 ⁇ l TBST. Phosphorylation is detected by adding 200 ⁇ l of SHP2-GFP TBST/BSA and incubating for 1 hour at RT with gentle rocking.
- Bound ZAPGFP is detected by reading the fluorescence of GFP using excitation at 485nm and measuring emission at 520nm. Wells containing no Src are used as a control ( Figure 10).
- the immobilised assay was also used to determine an inhibition curve for staurosporine, a general protein kinase inhibitor.
- the kinase assay was performed as above, using 2 unit of Src and incubating the enzyme with the immobilised SHPS-1 peptide for 90 minutes at 37°C. The results are shown in Figure 13.
- Example 20 Detection of Src kinase activity using an immobilised assay with a natural binding partner labelled with a coiled-coil tag and a fluorescent detector molecule (ZAP70-FJ-fluorescein peptide).
- Src kinase activity can be detected by the phosphorylation of a peptide derived from the T-cell receptor (TCR) ⁇ chain.
- TCR T-cell receptor
- the peptide is immobilised via a biotin/streptavidin interaction, and phosphorylation is detected by the binding of a fluorescein labelled SH2 domain from the ZAP70 kinase (ZAP70-FJ-fluorescein).
- the ZAP70SH2 domain is constructed as a fusion protein with a coiled-coil peptide based on the Fos/Jun coiled-coil peptide. Oligonucleotides based on the coiled-coil domain of Fos/Jun are designed and synthesised:
- the primers are annealed together by heating to 96 °C followed by slow cooling to room temperature.
- Complete double stranded DNA is generated by "filling in” the single stranded 5' overhangs using Klenow fragment of DNA polymerase I (NEB).
- the DNA fragment is purified by electrophoresis in 1.2% agarose gel and DNA is extracted from an isolated gel band using Qiagen spin columns. The purified fragment is digested with Sacl and Xhol and purified as above prior to ligation into the bacterial expression vector pET28a to generate vector FS101.
- FS101 is then digested with N/zel and EcoRI and D ⁇ A encoding ZAP70SH2 domain (from FS44) is ligated into this plasmid to generate vector FS102 that is designed to express ZAP70-Fos/Jun.
- ZAP70-FJ purification and labelling with fluorescein is then digested with N/zel and EcoRI and D ⁇ A encoding ZAP70SH2 domain (from FS44) is ligated into this plasmid to generate vector FS102 that is designed to express ZAP70-Fos/Jun.
- Fresh transformants of ZAP70-FJ pET-28a in BRL(DE3) is used to inoculate 3ml LB/kanamycin (lOO ⁇ g/ml).
- the starter cultures are incubated overnight at 37°C with shaking. From these starter culturess lml is used to inoculate 400ml TB/kanamycin (lOO ⁇ g/ml) in a 2L, baffled flask. Cultures are incubated at 37°C at 200 ⁇ m for approximately 5hrs until the O.D.600nm has reached 0.5Abs units. At this point cultures are induced by adding IPTG to a concentration of ImM. The cultures are then left incubating at room temperature for 2 hours with gentle shaking on a benchtop rotator.
- Bacteria are harvested by centrifugation at 3000 ⁇ m for 20mins.
- the bacterial pellet is resuspended in 25ml lysis buffer (50mM Pi pH 7.0, 300mM NaCl, 2% Proteinase inhibitor cocktail, 0.75mg/ml Lysozyme). Lysis of the resuspended cells is initiated by gentle stirring on a stirrer plate for lhr at room temperature.
- the partially lysed mixture is subjected to 2 cycles of freeze thawing in liquid nitrogen.
- the cells are sonicated on ice using a 10mm probe at high power. Sonication is performed on a pulse setting for a period of 3 min.
- Hexa-His-tagged proteins are purified from the clear lysate using TALON® resin (Clontech). Proteins are bound to the resin in a batchwise manner by gentle shaking at room temperature for 30 min. Non-HIS-tagged proteins are removed by washing the resin at least twice with lOx bed volume of wash buffer (50mM Pi pH 7.0, 300mM NaCl, 5mM fluorescence-blank Imidazole ).
- the washed resin is loaded into a 2 ml column and the bound proteins released with elution buffer (50mM Pi pH 7.0, 300mM NaCl, 150mM florescence-blank Imidazole). Elution is normally achieved after the first 0.5ml and within 2-3ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10%) glycerol.
- elution buffer 50mM Pi pH 7.0, 300mM NaCl, 150mM florescence-blank Imidazole. Elution is normally achieved after the first 0.5ml and within 2-3ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10%) glycerol.
- Peptide domains can be specifically labelled on amine or thiol groups with chemical fluorophores such as fluorescein or rhodamine. Fluorophores with thiol or amine reactive chemistries are readily available from commercial sources such as Molecular Probes. These fluorophores can be conjugated to peptides under mild conditions (e.g. 20 mM TES pH 7 for thiol directed labeling, or 200 mM sodium bicarbonate pH 8.3 for amine directed labeling, using 230 ⁇ M peptide in the presence of 200 ⁇ M label).
- Peptide FJ is labelled with fluorescein through amine directed labeling.
- Purified ZAP70-FJ is mixed with peptide 1 and the mixture monitored by FP to detect coiled-coil formation, resulting in ZAP70-FJ labelled with fluorescein.
- the peptides can be biotinylated under mild conditions using amine or thiol directed chemistry (e.g. 20 mM TES pH 7 for thiol directed labeling, and 200 mM sodium bicarbonate pH8.3 for amine directed labeling, using 230 ⁇ M peptide in the presence of 200 ⁇ M label).
- amine or thiol directed chemistry e.g. 20 mM TES pH 7 for thiol directed labeling, and 200 mM sodium bicarbonate pH8.3 for amine directed labeling, using 230 ⁇ M peptide in the presence of 200 ⁇ M label).
- Biotinylated peptides 1 and 2 are immobilised using 200 ⁇ l of 1 ug/ml peptide per well for 1 hour at room temperature in TBS containing 0.2%> Tween 20 (TBST) and 1% BSA.
- Src assay Peptide 2 bound to the plate is then treated with Src kinase (0.75 units) for 45 minutes at 37°C in kinase buffer (20 mM Tris-HCl pH7.2, 0.05 mM ATP, 10 mM MgCl 2 , 0.006% (v/v) Brij 35, 0.02 mg/ml BSA, 0.04% (v/v) ⁇ -mercaptoethanol).
- Enzyme is washed away using TBST/BSA and phosphorylation detected with 70 ⁇ l of a 1 in 10 dilution (in TBST + 1%) BSA) of fluorescein labelled ZAP70-FJ.
- Example 21 Detection of Src kinase activity using an immobilised assay with a natural binding partner labelled with GFP and an immobilised natural protein substrate.
- This assay comprises the immobilisation of TCR-4HA to a NeutrAvidin plate via a biotinylated 5HB peptide.
- the TCR chain is then phosphorylated by Src kinase. Phosphorylation is detected using ZAP-GFP.
- This DNA contains Nbel and Clal sites for cloning pu ⁇ oses and encodes the corresponding polypeptide sequence:
- Polypeptide 4HA YKGIAQLEQEIAQLEQE ⁇ AQLEQEIAQLEQE (SEQID ⁇ O:37).
- the primers are annealed together by heating to 96°C followed by slow cooling to room temperature.
- Complete double stranded DNA was generated by "filling in” the single stranded 5' overhangs using Klenow fragment of DNA polymerase I (NEB).
- the DNA fragment is purified by electrophoresis in 1.2% agarose gel and DNA is extracted from an isolated gel band using Qiagen spin columns.
- the purified fragment is digested with Nbel and Clal and purified as above prior to ligation into the plasmid FS19 to generate vector FS103.
- FS103 is then digested with NM and R ⁇ mHI and D ⁇ A encoding the 4 ⁇ A-TCR is ligated into pET28a to generate a bacterial expression vector FS104.
- Fresh transformants of Zap70-FJ pET-28a in BRL(DE3) and 4HA-TCR pET-28a in BRL(DE3) pLysS are used to inoculate 3 ml LB/kanamycin (100 g/ml).
- the starter cultures are incubated overnight at 37°C with shaking. From these starter cultures 1 ml is used to inoculate 400 ml TB/kanamycin (100 g/ml) in a 2L, baffled flask. Cultures are incubated at 37°C at 200 ⁇ m for approximately 5 hrs until the O.D. 600 nm reaches 0.5 Abs units. At this point cultures are induced by adding IPTG to a concentration of 1 mM. The cultures are then left incubating at room temperature overnight with gentle shaking on a benchtop shaker.
- Bacteria are harvested by centrifugation at 3000 ⁇ m for 20mins.
- the bacterial pellet is resuspended in 25 ml lysis buffer (50 mM Pi pH 7.0, 300 mM ⁇ aCl, 2% Proteinase inhibitor cocktail, 0.75 mg/ml Lysozyme). Lysis of the resuspended cells is initiated by gentle stirring on a stirrer plate for lhr at room temperature.
- the partially lysed mixture is subjected to 2 cycles of freeze thawing in liquid nitrogen.
- the cells are sonicated on ice using a 10mm probe at high power. Sonication is performed on a pulse setting for a period of 3 min.
- HIS tagged proteins were purified from the clear lysate using Talon resin (Clontech). Proteins are bound to the resin in a batchwise manner by gentle shaking at room temperature for 30 min. Non-HIS tagged proteins are removed by washing the resin at least twice with lOx bed volume of wash buffer (50 mM Pi pH 7.0, 300 mM NaCl, 5mM fluorescence-blank imidazole).
- the washed resin is loaded into a 2 ml column and the bound proteins released with elution buffer (50 mM Pi pH 7.0, 300 mM NaCl, 150mM florescence-blank imidazole). Elution is normally achieved after the first 0.5 ml and within 2-3 ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10% glycerol.
- elution buffer 50 mM Pi pH 7.0, 300 mM NaCl, 150mM florescence-blank imidazole. Elution is normally achieved after the first 0.5 ml and within 2-3 ml in total. Proteins are stored at -80°C after snap freezing in liquid nitrogen in the presence of 10% glycerol.
- Biotinylation of the 5HB peptide is performed by preparing a 200 ⁇ l solution of 220 ⁇ M (1 mg/ml) 5HB in 20 mM TES pH 7.0. Thiol directed Biotin BMCC 8 ⁇ l (5 mg/ml) is added to this making the final concentration of Biotin 200 ⁇ M. The solution is mixed at room temperature for 2 hrs wrapped in tin foil.
- the biotinylated 5HB peptide is then bound to a black Reacti-Bind, Neutravidin plate (Pierce).
- the 5HB-Biotin stock is diluted tol ⁇ g/ml in TBST/BSA.
- a 200 ⁇ l volume is then added to the wells of the plate.
- the plate is incubated at room temperature for 1 hr with gentle shaking.
- the wells are washed with 3x 200 ⁇ l of TBST/BSA. Where 5HB free controls are required the wells are incubated in TBST/BSA only.
- TCR-4HA construct is then bound to the 5HB peptide.
- Increasing volumes (2, 5, 10, 20 ⁇ l) of TCR-4HA are diluted into 200 ⁇ l of TBST/BSA and added to the 5HB-Biotin wells. Again, where TCR-4HA free controls are required, the wells are incubated in TBST/BSA. The plate is incubated at room temperature for 1 hr with gentle shaking. The wells are washed in 3x 200 ⁇ l of TBST/BSA.
- Phosphorylation of immobilised TCR-4HA is performed by adding 200 ⁇ l of phosphorylation mix to the wells.
- the mix consists of 40 ⁇ l ATP mix, 40 ⁇ l kinase dilution buffer, 120 ⁇ l H 2 O and 1 unit Src kinase. Note that where non-phosphorylated TCR-4HA controls are required no Src kinase is added.
- the plate is incubated at 37°C for 1/z hours.
- the wells are washed with 3x 200 ⁇ l TBST/BSA.
- the ZAP-GFP construct is used as a probe in this assay. Phosphorylation-dependent binding of the two natural binding domains results in retention of GFP on the plate. The associated green fluorescent signal is detected on a fluorescent plate reader.
- a lOOx dilution of ZAP-GFP ( ⁇ 1 ⁇ M) is prepared in TBST/BSA. To each well 200 ⁇ l of this solution is added and incubated for 1 hr at room temperature with gentle shaking. The wells are washed with 3x 200 ⁇ l TBST/BSA.
- the bar chart ( Figure 15) shows that the GFP signal with phosphorylated TCR-4HA is 3-6x greater than that for the de-phosphorylated construct. Non-specific signals are low, as indicated by 5HB peptide only and buffer only control experiments. It is also notable that GFP can give a 2 fold increase in signal over antibody detection. This experiment has been repeated in triplicate where a single concentration of TCR-4HA (7.5 ⁇ l) is used. Alongside this an inhibition experiment was performed. Staurosporine (3 ⁇ l of a 1 mM stock) is added to 3 wells at the phosphorylation stage. This gives each well a final staurosporine concentration of 15 ⁇ M. The bar chart ( Figure 16) shows that the assay is reproducible and that the presence of staurosporine kills off any Src kinase activity. Again the antibody controls have a much poorer signal but confirm the ZAP-GFP data.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00906474A EP1163526A2 (en) | 1999-02-25 | 2000-02-25 | High throughput assay based on the use of a polypeptide binding pair |
AU28138/00A AU2813800A (en) | 1999-02-25 | 2000-02-25 | High throughput assay |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9904398.6 | 1999-02-25 | ||
GBGB9904398.6A GB9904398D0 (en) | 1999-02-25 | 1999-02-25 | High throughput assay |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2000050902A2 true WO2000050902A2 (en) | 2000-08-31 |
WO2000050902A3 WO2000050902A3 (en) | 2000-12-14 |
Family
ID=10848521
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2000/000669 WO2000050902A2 (en) | 1999-02-25 | 2000-02-25 | High throughput assay based on the use of a polypeptide binding pair |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1163526A2 (en) |
AU (1) | AU2813800A (en) |
GB (1) | GB9904398D0 (en) |
WO (1) | WO2000050902A2 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003050138A2 (en) * | 2001-12-12 | 2003-06-19 | Darcy Birse | Method of modification of a protein or polypeptide |
US6791960B1 (en) | 1999-03-15 | 2004-09-14 | Lg Information And Communications, Ltd. | Pilot signals for synchronization and/or channel estimation |
US7496132B2 (en) | 1999-03-15 | 2009-02-24 | Kg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
US7616681B2 (en) | 1999-03-15 | 2009-11-10 | Lg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
US7643540B2 (en) | 1999-03-15 | 2010-01-05 | Lg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0392808A2 (en) * | 1989-04-10 | 1990-10-17 | The University Of Georgia Research Foundation, Inc. | Enzyme luminescence assay |
WO1996020211A1 (en) * | 1994-12-23 | 1996-07-04 | Ludwig Institute For Cancer Research | Phosphotyrosine assay and peptides for use therein |
WO1996021040A1 (en) * | 1994-12-29 | 1996-07-11 | Research Foundation Of State University Of New York | Rapid assay of activators and inhibitors of clotting |
WO1996035806A1 (en) * | 1995-05-12 | 1996-11-14 | Schering Corporation | Surface plasmon resonance based enzymatic assay |
WO1998020024A1 (en) * | 1996-11-04 | 1998-05-14 | Merck Frosst Canada & Co. | Ligands for phosphatase binding assay |
WO1998039471A1 (en) * | 1997-03-07 | 1998-09-11 | Tropix, Inc. | Protease inhibtor assay |
WO1999011774A1 (en) * | 1997-08-30 | 1999-03-11 | Fluorescience Limited | Polypeptides comprising a coiled-coil and an addition site |
-
1999
- 1999-02-25 GB GBGB9904398.6A patent/GB9904398D0/en not_active Ceased
-
2000
- 2000-02-25 AU AU28138/00A patent/AU2813800A/en not_active Abandoned
- 2000-02-25 WO PCT/GB2000/000669 patent/WO2000050902A2/en not_active Application Discontinuation
- 2000-02-25 EP EP00906474A patent/EP1163526A2/en not_active Withdrawn
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0392808A2 (en) * | 1989-04-10 | 1990-10-17 | The University Of Georgia Research Foundation, Inc. | Enzyme luminescence assay |
WO1996020211A1 (en) * | 1994-12-23 | 1996-07-04 | Ludwig Institute For Cancer Research | Phosphotyrosine assay and peptides for use therein |
WO1996021040A1 (en) * | 1994-12-29 | 1996-07-11 | Research Foundation Of State University Of New York | Rapid assay of activators and inhibitors of clotting |
WO1996035806A1 (en) * | 1995-05-12 | 1996-11-14 | Schering Corporation | Surface plasmon resonance based enzymatic assay |
WO1998020024A1 (en) * | 1996-11-04 | 1998-05-14 | Merck Frosst Canada & Co. | Ligands for phosphatase binding assay |
WO1998039471A1 (en) * | 1997-03-07 | 1998-09-11 | Tropix, Inc. | Protease inhibtor assay |
WO1999011774A1 (en) * | 1997-08-30 | 1999-03-11 | Fluorescience Limited | Polypeptides comprising a coiled-coil and an addition site |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6791960B1 (en) | 1999-03-15 | 2004-09-14 | Lg Information And Communications, Ltd. | Pilot signals for synchronization and/or channel estimation |
US7496132B2 (en) | 1999-03-15 | 2009-02-24 | Kg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
US7602841B2 (en) | 1999-03-15 | 2009-10-13 | Lg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
US7616681B2 (en) | 1999-03-15 | 2009-11-10 | Lg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
US7643540B2 (en) | 1999-03-15 | 2010-01-05 | Lg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
US7848393B2 (en) | 1999-03-15 | 2010-12-07 | Lg Electronics Inc. | Pilot signals for synchronization and/or channel estimation |
WO2003050138A2 (en) * | 2001-12-12 | 2003-06-19 | Darcy Birse | Method of modification of a protein or polypeptide |
WO2003050138A3 (en) * | 2001-12-12 | 2003-12-24 | Darcy Birse | Method of modification of a protein or polypeptide |
Also Published As
Publication number | Publication date |
---|---|
EP1163526A2 (en) | 2001-12-19 |
AU2813800A (en) | 2000-09-14 |
GB9904398D0 (en) | 1999-04-21 |
WO2000050902A3 (en) | 2000-12-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7060506B2 (en) | Compositions and methods for monitoring the modification of modification dependent binding partner polypeptides | |
US6972198B2 (en) | Methods and compositions using protein binding partners | |
ES2926463T3 (en) | Activation of bioluminescence by structural complementation | |
US6900304B2 (en) | Emission ratiometric indicators of phosphorylation | |
US7166475B2 (en) | Compositions and methods for monitoring the modification state of a pair of polypeptides | |
US8669074B2 (en) | Chimeric phosphorylation indicator | |
AU2002312149A1 (en) | Emission ratiometric indicators of phosphorylation | |
US20060148017A1 (en) | Compositions and methods for monitoring enzyme activity | |
US6828106B2 (en) | Methods and compositions using coiled binding partners | |
US6410687B1 (en) | Polypeptides for the detection of microtubule depolymerization inhibitors | |
US8323919B2 (en) | Assay methods for identifying glycogen synthase kinase 3 modulators | |
EP1163363B1 (en) | Compositions and methods for monitoring the modification of natural binding partners | |
EP1171627B1 (en) | Assay for measuring different enzyme activities simultaneously | |
EP1163526A2 (en) | High throughput assay based on the use of a polypeptide binding pair | |
US6972182B1 (en) | Methods and compositions using coiled binding partners | |
EP1155321A1 (en) | Compositions and methods for monitoring the modification of engineered binding partners | |
WO2000050635A1 (en) | Methods and compositions using binding partners | |
US20040161784A1 (en) | Assays for the detection of microtubule depolymerization inhibitors | |
US20060040319A1 (en) | Multiple sensor-containing active modified polypeptides, preparation and uses thereof | |
US7105307B2 (en) | Compositions and methods for screening for modulators of enzymatic activity | |
EP1155326A1 (en) | Protein assay | |
WO2014179494A1 (en) | Sensors and assays for ubiquitin or ubiquitin-like proteins |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000906474 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2000906474 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2000906474 Country of ref document: EP |