CA2265935C - Heat shock protein complexes - Google Patents

Heat shock protein complexes Download PDF

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CA2265935C
CA2265935C CA002265935A CA2265935A CA2265935C CA 2265935 C CA2265935 C CA 2265935C CA 002265935 A CA002265935 A CA 002265935A CA 2265935 A CA2265935 A CA 2265935A CA 2265935 C CA2265935 C CA 2265935C
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heat shock
shock protein
adp
proteins
column
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CA2265935A1 (en
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Erik S. Wallen
Jan Roigas
Pope L. Moseley
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UNM Rainforest Innovations
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STC UNM
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Priority claimed from US08/717,239 external-priority patent/US5747332A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B5/00—Optical elements other than lenses
    • G02B5/30—Polarising elements
    • G02B5/3025—Polarisers, i.e. arrangements capable of producing a definite output polarisation state from an unpolarised input state
    • G02B5/3033—Polarisers, i.e. arrangements capable of producing a definite output polarisation state from an unpolarised input state in the form of a thin sheet or foil, e.g. Polaroid
    • G02B5/3041—Polarisers, i.e. arrangements capable of producing a definite output polarisation state from an unpolarised input state in the form of a thin sheet or foil, e.g. Polaroid comprising multiple thin layers, e.g. multilayer stacks
    • G02B5/305—Polarisers, i.e. arrangements capable of producing a definite output polarisation state from an unpolarised input state in the form of a thin sheet or foil, e.g. Polaroid comprising multiple thin layers, e.g. multilayer stacks including organic materials, e.g. polymeric layers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/803—Physical recovery methods, e.g. chromatography, grinding

Abstract

A method for purifying heat shock protein complexes is provided which comprises the steps of adding a solution containing heat shock protein complexes, in which heat shock proteins are associated with peptides, polypeptides, denatured proteins or antigens, to a column containing an ADP matrix to bind the heat shock protein complexes to the ADP matrix and adding a buffer containing ADP to the column to remove the heat shock protein complexes in an elution product. Additionally a method for synthesizing heat shock protein complexes and purifying the complexes so produced is provided which comprise s the steps of adding heat shock proteins to an ADP matrix column to bind them to the matrix, adding a solution of peptides, polypeptides, denatured proteins or antigens to the column to bind them to the heat shock protein as heat shock protein complexes and adding a buffe r containing ADP to the column to remove the complexes in an elution product.

Description

1015202530CA 02265935 2004-03-29HEAT SHOCK PROTEIN COMPLEXESBACKGROUND OF THE INVENTIONField of the InventionThe present invention relates generally to methods for purifying andsynthesizing heat shock protein complexes.Descrimion of the Prior ArtHeat shock proteins (HSPS) are associated in cells with a broad spectrum ofpeptides, polypeptides, denatured proteins and antigens with which they formcomplexes. Such HSP-peptide complexes have been described as being useful invaccines against cancers and infectious diseases by Srivastava et al., “Heat shockprotein-peptide complexes in cancer immunotherapy” in Current Opinion inImmunology (1994), 6:728—732; Srivastava, “Peptide-Binding Heat Shock Proteins inthe Endoplasmic Reticulum” in Advances in Cancer Research (1993), 62:153-177.The HSP-peptide complexes appear to work as Vaccines, because they may function asantigen carrying and presentation molecules. The development of vaccines using suchantigens has been described by Baltz, “Vaccines in the treatment of Cancer” in Am. J.Health-Syst. Pharm. (1995), 52:2574-2585. The antigenicity of heat shock proteinsappears to derive not from the heat shock protein itself, but from the associatedpeptides, see Udono et al., “Heat Shock Protein 70-associated Peptides Elicit SpecificCancer Immunity” in J. Exp. Med. (1993), 178:139l-1396; Srivastava et al., “Heatshock proteins transfer peptides during antigen processing and CTL priming” inImmunogenetics (1994), 39:93-98; Srivastava, “A Critical Contemplation on the Rolesof Heat Shock Proteins in Transfer of Antigenic Peptides During AntigenPresentation” in Behring Inst. Mitt. (1994), 94:37-47. HSPs appear to be part of theprocess by which peptides are transported to the Major Histocompatibility Complex(MHC) molecules for surface presentation.11015202530CA 02265935 2004-03-29A number of different HSPs have been shown to exhibit immunogenicity including:gp96, hsp90 and hsp70, see Udono et al., supra. and Udono et al., “Comparison of Tumor-Specific Immunogenicities of Stress-Induced Proteins gp96, hsp90, and hsp 70" in Journal ofImmunology (1994), 5398-5403; gp96 and grp94, Li et al., “Tumor rejection antigengp96/grp94 is an ATPase: implications for protein folding and antigen presentation” in TheEMBO Journal, Vol. 12, No. 8 (1993), 3143-3151; and gp96, hsp90 and hsp70, Blachere etal., “Heat Shock Protein Vaccines Against Cancer” in Journal Of Immunotherapy (1993),14:352-356.Heat shock proteins have been purified using a procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose, see Welchet al., “Rapid Purification of Mammalian 70,000-Dalton Stress Proteins: Affinity of theProteins for Nucleotides” in Molecular ‘and Cellular Biology (June 1985), 1229-1237.However, previous methods of purifying HSPS such as this one purify the heat shock proteinswithout the associated peptides. Other methods that do purify HSPS together with theirassociated peptides are complicated and expensive.SUMMARY OF THE INVENTIONTherefore, it is an object of the invention to provide a simple and inexpensive methodfor purifying heat shock proteins together with their associated peptides. polypeptides,denatured proteins or antigens from cell lysates.It is a further object of the invention to provide a method for synthesizing heat shockprotein complexes that is capable of forming these complexes from heat shock proteins andpeptides, polypeptides, denatured proteins or antigens from different cells and from differentspecies.The present invention provides a method for purifying heat shock protein complexescomprising the steps of adding a solution containing heat shock protein complexes, in which1015202530CA 02265935 2004-03-29heat shock proteins are associated with peptides, polypeptides, denatured proteins orantigens, to a column containing an ADP matrix to bind the heat shock proteinscomplexes to the ADP matrix and then adding a buffer containing ADP to the column toremove the heat shock protein complexes in an elution product, with a proviso that theheat shock proteins in the heat shock protein complexes do not consist solely of eukaryotehsp70.The present invention also provides a method for synthesizing heat shock proteincomplexes by adding heat shock proteins to an ADP matrix column to bind them to thematrix, adding a complexing solution of peptides, polypeptides, denatured proteins orantigens to the column to bind them to the heat shock proteins as heat shock proteincomplexes and then adding a buffer containing ADP to the column to remove thecomplexes in an elution product. The methods of this invention may also be used topurify heat shock protein complexes so synthesized.The methods of this invention may further comprises the step of adding apurifying buffer solution to the column to elute proteins that do not bind with the ADP .matrix. A purifying buffer solution may comprise at least one member of the groupconsisting of GTP and non-adenosine containing nucleotides.In the methods of this invention, solutions containing heat shock proteincomplexes and complexing solutions may be from cell lysates, membrane isolates, andprotease treated cell lysates.This invention also provides a purified ADP-heat shock protein-peptide complex,wherein the heat shock protein is selected from the group consisting of: eukaryote hsp65,rubisco binding protein and TCP-1; and, yeast Mif4, TCPalpha and TCPbeta.This invention also provides a purified ADP-heat shock protein-peptide complex,wherein the heat shock protein is selected from the group consisting of: hsp104, hsp105and hspl 10.This invention also provides a purified ADP-heat shock protein-peptide complex,wherein the heat shock protein is selected from the group consisting of: hsp90, gp96, andg1p94.This invention also provides a purified ADP-heat shock protein-peptide complex,wherein the heat shock protein is selected from the group consisting of: yeast Ssa, Ssb,and Ssc.10152025CA 02265935 2004-03-29This invention also provides a purified ADP-heat shock protein-peptide complex,wherein the heat shock protein is eukaryote Grp75.This invention also provides a purified ADP-heat shock protein-peptide complex,wherein a heat shock protein-peptide portion of said ADP-heat shock protein-peptidecomplex comprises a non-naturally occurring heat shock protein-peptide combination,excluding the combination of DnaK and NCA-SNase.BRIEF DESCRIPTION OF THE DRAVVIN GSFigure 1 is a drawing of a western blot of fractions taken from a purification using theADP purification matrix;Figure 2 is a plot of HPLC data of material treated with NaCl after being purified bythe method of the invention and filtered through a 20,000 molecular weight cut-off filter; andFigure 3 is a plot of HPLC data of material treated with ATP after being purified bythe method of the invention and filtered through a 20,000 molecular weight cut-off filter.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTIn one preferred embodiment, the present invention provides a method for isolatingheat shock protein complexes from a solution containing heat shock proteins using an ADP3a1015202530CA 02265935 2004-03-29matrix. Each of the heat shock protein complexes consists of a heat shock protein (HSP) thatis bound tightly to an incomplete protein in a cell.According to the method of the invention, solutions containing these HSP complexesare added to a conventional column, such as an agarose gel column, to which ADP has beenadded to fonn an ADP matrix. Suitable ADP-agarose columns include those described in U.SPatent Nos. 5,114,852; 5,268,465; 5l32,407; and 5,541,095.ADP has a strong affinity fortheHSP complexes and unlike ATP, does not break down the HSP complexes when it binds tothem.Typically the solution from which the heat shock protein complexes are purified is acell lysate from a tumor in which the HSPs are already present. However, the inventioncontemplates that the solution containing HSP complexes to be purified may be produced bymixing an already purified heat shock protein with a cell lysate, a membrane isolate(materials isolated from a cell membrane) or a protease treated cell lysate containing peptides,polypeptides, denatured proteins to ‘produce a solution of HSP complexes. For the purposes ofthe present invention the term “peptides” refers to all peptides and polypeptides includingdenatured proteins, and recombinant or otherwise purified tumor or infectious diseaseantigens that may be associated with heat shock proteins, either naturally or synthetically.In order to increase the number of heat shock proteins in the solution added to theADP matrix column, the solution may be incubated at a temperature of 37 to 50°C. andadditional ADP may be added to the solution prior to adding it to the column. If the HSPcomplex solution is a cell lysate, additional HSPS may be added to the lysate to formadditional complexes.A buffer solution containing ADP is added to the column to elute the HSP complexesfrom the ADP matrix as an elution product containing the HSP complexes. In addition toADP, this buffer solution may also contain small amounts of components such as sodiumchloride that aid in the removal of the complexes from the ADP matrix.1015202530CA 02265935 2004-03-29In order to produce a more purified elution product, after the HSP complexes havebeen bound to the ADP matrix, a purifying buffer solution may be added to the column toelute other proteins loosely bound to the matrix. This purifying buffer solution preferablycontains GTP or another non-adenosine containing nucleotideThe method of the invention takes advantage of the fact that HSPs are associated withpeptides inside the cell. This purification method maintains the HSP-peptide associationnecessary to develop vaccines or immunotherapeutic tools for tumors and for infectiousdiseases since HSPS have not been shown to be helpful as antigens without the associatedpeptides.In another embodiment the invention provides a method for synthesizing HSPcomplexes and purifying the complexes so produced. In this method, purified HSPS arebound to an ADP matrix column. Then a preparation of peptides, polypeptides, denaturedproteins and/or antigens is added to, an ADP matrix column to bind to the HSPS in the matrix.The method then proceeds similarly to the first embodiment of the invention. A buffersolution containing ADP is added to the column to elute the HSP complexes from the ADPmatrix as an elution product containing the HSP complexes. This buffer solution may containsmall amounts of components such as sodium chloride that aid in the removal of thecomplexes from the ADP matrix.As with the first embodiment of the invention, a purifying buffer solution containingGTP or another non-adenosine containing nucleotide may be added to the column to eluteother proteins loosely bound to the matrix.This second embodiment permits HSP complexes to be formed from HSPs andpeptides, denatured proteins or antigens from different cells or even different species.Although there are many heat shock proteins that may be used in the method of thepresent invention, heat shock proteins that have proven particularly useful include membersof the hsp6O family, hsp70 family, hsp9O family and the hsp104-105 family.1015202530CA 02265935 2004-03-29Members of the hsp60 family include hsp60, hsp65, rubisco binding protein, andTCP-1 in eukaryotes; and GroE1/GroES in prokaryotes; Mif4, and TCPlalpha and beta inyeast.Members of the hsp7O family include DnaK proteins from prokaryotes, Ssa, Ssb, andSsc from yeast, hsp70, Grp75 and G1p78(Bip) from eukaryotes. Figure l is a drawing of awestern blot of fractions taken from a purification using the method of the invention. Theelution was started at fraction #10 and hsp7O protein appears in fraction #14.Members of the hsp90 family include hsp90, g96 and grp94.Members of the hsp104-105 family include hspl 05 and hspl 10.The HSP/peptide complexes are eluted from the matrix using an ADP containingbuffer. It also helps I-ISPS to be added to peptide mixtures and the complexes for use as avaccine.The invention will now be described by way of example. The following examples areillustrative and are not meant to limit the scope of the invention which is set forth by theappointed claims.EXAMPLE 1A confluent T-75 of B16-F 1 mouse melanoma cells was rinsed 3x with PBS. 1 ml ofPBS was added and the cells were scraped to create a suspension. The suspension was spunfor 5 minutes at 1000rpm to pellet the cells. The supernatant was removed and the cellsresuspended in 1.5 ml of a hypotonic buffer (30mM NaHCO3, pH 7.1). The suspension wastransferred to a glass tube and the cells were lysed with a Teflon® pestle and power drill. Thelysate was transferred to a microcentrifuge tube and spun at high speed to pellet theundissolved fraction. Total protein of the lysate was determined using the Bradford method.Solution containing 100p.g of total protein was brought up to 300p] total volume with the10CA 02265935 2004-03-29addition of Phosphate buffer (O.lM KH2P04, 10 mM NaCl, lmM EDTA, pH 7.2) and thesolution was added to a 5 ml ADP-agarose column (linked through C-8, Sigma ChemicalCo.) and allowed to run into the column with 5 ml of Phosphate‘ buffer and then buffer B(20mM TRIS, 20mM NaCl, 15 mM EDTA, 15mM Beta-mercaptoethanol, pH 7.5) with60mM ADP was added at the start of fraction 10 to elute the complexes. After completion ofthe run, 50p.l of each fraction was run onto a 7.5% SDS PAGE gel, transferred tonitrocellulose, probed with an antibody for the inducible and constitutive hsp70 (N27,Stressgen Biotechnologies), and then a secondary alkaline phosphate linked antibody. A blotwas developed in a buffer containing BCIP and NBT. A drawing of this plot is shown inFigure 1.101520CA 02265935 2004-03-29EXAMPLE 2PC-3 lysate was run over a agarose column containing an ADP matrix according themethod of the invention. The HSP containing fraction was then eluted with ADP. The elutedfraction containing HSPS was filtered using a 20,000 molecular weight cut-off (MWC) filterand several rinses of buffer A (25mMTris, 20mM Hepes, 47.5mM KCl, and 2.25mMMg(OAc)2, pH 7.2) were applied. The sample was split into two microcentrifuge tubes andeither ATP (to 10mM) or NaCl (to lmM) was added. The tubes were then incubatedovernight at 37°C. Each solution was then spun through a 20,000MWC filter and the filteredmaterial was applied to an HPLC column. The HPLC was accomplished using a C18 reversephase column (Vydacf 201TB54). The starting buffer was 0.1% TF A in dH,0 and the materialwas eluted using a gradient of 0.1% TFA in ACN. Figure 2 shows HPLC data for the materialtreated with NaCl after being purified with the ADP matrix and filtered through the 20,000molecular weight cut-off filter. Figure 3 shows the HPLC data for the material treated withATP after being purified with the ADP matrix and filtered through the 20.000 molecularweight cut-off filter. The HPLC data in Figures 2 and 3 is consistent with the data for hsp70described in Udono e/ 511., “Heat Shock Protein 70-associated Peptides Elicit Specific CancerImmunity” in./. Exp. Med. (1993), l78:l39l-1396.Although the present invention has been fully described in conjunction with thepreferred embodiment thereof with reference to the accompanying drawings, it is to beunderstood that various changes and modifications may be apparent to those skilled in the art.Such changes and modifications are to be understood as included within the scope of thepresent invention as defined by the appended claims, unless they depart therefrom.*Trade-mark

Claims (36)

WHAT IS CLAIMED
1. A method for purifying heat shock protein complexes comprising the steps of:
adding a solution containing heat shock protein complexes in which one or more heat shock proteins are associated with at least one member of the group consisting of peptides, polypeptides, denatured proteins and antigens, to a column containing an ADP
matrix to bind the heat shock protein complex to the ADP matrix; and adding a buffer containing ADP to the column to remove the heat shock protein complexes in an elution product, with a proviso that the heat shock proteins in the heat shock protein complexes do not consist solely of eukaryote hsp70.
2. The method of claim 1 further comprising the step of adding a purifying buffer comprising at least one member of the group consisting of GTP and non-adenosine containing nucleotides to the column to elute proteins other than heat shock proteins that are loosely bound to the ADP matrix.
3. The method of claim 1 further comprising the step of adding a buffer containing GTP to the column to elute proteins other than heat shock proteins that are loosely bound to the matrix.
4. The method of claim 1, 2, or 3, wherein the solution containing heat shock protein complexes comprises a cell lysate.
5. The method of any one of claims 1-4, further comprising the step of incubating a solution in which one or more heat shock proteins and at least one member of the group consisting of peptides, polypeptides, denatured proteins and antigens are present, at a temperature of 37 to 50° C, to induce the one or more heat shock proteins to bind to the at least one member, thereby forming the solution containing heat shock protein complexes.
6. The method of any one of claims 1-5, further comprising the step of adding ADP
to the solution containing heat shock protein complexes prior to adding the solution to the column to induce heat shock proteins present in the solution to bind to peptides, polypeptides, denatured proteins and antigens present in the solution to form heat shock protein complexes.
7. The method of any one of claims 1-6, further comprising adding purified heat shock proteins to the solution containing heat shock protein complexes, prior to adding the solution to the column.
8. The method of any one of claims 1-7, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of the group consisting of eukaryote hsp60, eukaryote hsp65, eukaryote rubisco binding protein, eukaryote TCP-1, yeast GroEL/GroES, yeast Mif4, yeast TCPalpha and yeast TCPbeta.
9. The method of any one of claims 1-7, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of the group consisting of: hsp104, hsp105 and hsp110.
10. The method of any one of claims 1-7, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of the group consisting of: prokaryote DnaK proteins; yeast Ssa, yeast Ssb, yeast Ssc, eukaryote Grp75 and eukaryote Grp78(Bip).
11. The method of any one of claims 1-7, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of the group consisting of: hsp90, gp96 and grp94.
12. The method of any one of claims 1-11, wherein a heat shock protein complex is formed by mixing a heat shock protein with a complexing agent selected from the group consisting of peptides, polypeptides, denatured proteins and antigens.
13. A method for synthesizing heat shock protein complexes comprising the steps of:
adding a solution containing a heat shock protein to a column containing an ADP
matrix to bind the heat shock protein to the ADP matrix;

adding a complexing solution comprising a complexing agent selected from the group consisting of peptides, polypeptides, denatured proteins and antigens, to the column to form heat shock protein complexes with the heat shock protein bound to the ADP
matrix; and adding a buffer containing ADP to the column to remove the heat shock protein complexes in an elution product.
14. The method of claim 13 further comprising the step of adding a purifying buffer comprising at least one member of the group consisting of GTP and non-adenosine containing nucleotides to the column to elute proteins other than heat shock proteins that are loosely bound to the ADP matrix.
15. The method of claim 13 further comprising the step of adding a buffer solution containing GTP to the column to elute proteins other than heat shock proteins that are loosely bound to the matrix.
16. The method of claim 13, 14, or 15, wherein the complexing solution comprises a peptide mixture selected from the group consisting of cell lysates, membrane isolates, and protease treated cell lysates.
17. The method of any one of claims 13-16, further comprising the step of incubating a solution containing heat shock protein and one or more members of the group consisting of peptides, polypeptides, denatured proteins and antigens, at a temperature of 37 to 50°
C to induce the heat shock protein present in the solution to bind to the at least one member present in the solution to form the heat shock protein solution added to the column.
18. The method of any one of claims 13-17, further comprising the step of adding ADP to the solution containing heat shock protein complexes prior to adding the solution to the column to induce heat shock proteins present in the solution to bind to peptides, polypeptides, denatured proteins and antigens present in the solution to form heat shock protein complexes.
19. The method of any one of claims 13-18, further comprising adding purified heat shock proteins to the solution containing heat shock proteins prior to adding the solution to the column.
20. The method of any one of claims 13-19, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of the group consisting of eukaryote hsp60, eukaryote hsp65, eukaryote rubisco binding protein, eukaryote TCP-l, yeast GroEL/GroES, yeast Mif4, yeast TCPalpha and yeast TCPbeta.
21. The method of any one of claims 13-19, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of one of the group consisting of: hsp104, hsp105 and hsp110.
22. The method of any one of claims 13-19, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of the group consisting of: prokaryote DnaK proteins; yeast Ssa, yeast Ssb, yeast Ssc, eukaryote Grp75 and eukaryote Grp78(Bip).
23. The method of any one of claims 13-19, wherein the heat shock protein complexes include complexes in which heat shock proteins are one or more members of the group consisting of: hsp90, gp96 and grp94.
24. A purified ADP-heat shock protein-peptide complex, wherein the heat shock protein is selected from the group consisting of: eukaryote hsp65, eukaryote rubisco binding protein, eukaryote TCP-1, yeast Mif4, yeast TCPalpha and yeast TCPbeta.
25. A purified ADP-heat shock protein-peptide complex, wherein the heat shock protein is selected from the group consisting of: hsp104, hsp105 and hsp110.
26. A purified ADP-heat shock protein-peptide complex, wherein the heat shock protein is selected from the group consisting of: hsp90, gp96, and grp94.
27. A purified ADP-heat shock protein-peptide complex, wherein the heat shock protein is selected from the group consisting of: yeast Ssa, Ssb, and Ssc.
28. A purified ADP-heat shock protein-peptide complex, wherein the heat shock protein is eukaryote Grp75.
29. The purified complex of any one of claims 24-28, wherein the peptide is a polypeptide or protein.
30. The purified complex of any one of claims 24-29, wherein a heat shock protein-peptide portion of said ADP-heat shock protein-peptide complex comprises a non-naturally occurring heat shock protein-peptide combination.
31. The ADP-heat shock protein-peptide complex of claim 30, wherein the heat shock protein-peptide portion of said complex comprises a heat shock protein from one cell of an individual and a peptide from a second cell of the individual.
32. The ADP-heat shock protein-peptide complex of claim 30, wherein the heat shock protein-peptide portion of said complex comprises a heat shock protein from a first individual and a peptide from a second individual.
33. The ADP-heat shock protein-peptide complex of claim 30, wherein the heat shock protein-peptide portion of said complex comprises a heat shock protein from a first organism and a peptide from a second organism.
34. The ADP-heat shock protein-peptide complex of claim 30, wherein the heat shock protein-peptide portion of said complex comprises a heat shock protein from one species and a peptide from a second species.
35. The ADP-heat shock protein-peptide complex of any one of claims 24-34, wherein the ADP-heat shock protein-peptide complex is purified by the steps of:

adding a heat shock protein complex comprising a non-naturally occurring combination of a heat shock protein associated with at least one member of the group consisting of peptides, polypeptides, denatured proteins and antigens, to a column containing an ADP matrix to bind the heat shock protein complex to the ADP
matrix; and adding a buffer containing ADP to the column to remove the heat shock protein complex in an elution product.
36. The ADP-heat shock protein-peptide complex of any one of claims 24-35, wherein said ADP-heat shock protein-peptide complex is synthesized by the steps of:
adding a heat shock protein to a column containing an ADP matrix to bind the heat shock protein to the ADP matrix;
adding to the column a complexing solution comprising a complexing agent which does not naturally occur in combination with the heat shock protein, said complexing agent being selected from the group consisting of peptides, polypeptides, denatured proteins and antigens, to form a heat shock protein complex with the heat shock protein bound to the ADP matrix; and adding a buffer containing ADP to the column to remove the heat shock protein complex in an elution product.
CA002265935A 1996-09-20 1997-09-19 Heat shock protein complexes Expired - Fee Related CA2265935C (en)

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Application Number Priority Date Filing Date Title
US08/717,239 US5747332A (en) 1996-09-20 1996-09-20 Methods for purifying and synthesizing heat shock protein complexes
US08/717,239 1996-09-20
US08/934,139 US6066716A (en) 1996-09-20 1997-09-19 Purified heat shock protein complexes
PCT/US1997/016937 WO1998012208A1 (en) 1996-09-20 1997-09-19 Heat shock protein complexes
US08/934,139 1997-09-19

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US5981706A (en) 1999-11-09
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