WO2003040312A2 - Multifunctional green fluorescent protein and uses thereof - Google Patents

Abstract

The present invention relates to a multifunctional green fluorescent protein engineered to have a purification tag inserted into a β-barrel structure of the green fluorescent protein molecule, and nucleic acid constructs, host cells, expression systems, and fusion proteins containing the multifunctional green fluorescent protein. The present invention also relates to microarrays having a tagged-green fluorescent protein. Also disclosed are methods for purifying a protein or polypeptide, identifying expression of a protein or polypeptide in biological material, and identifying a target molecule using a multifunctional green fluorescent protein of the present invention.

Description

MULTIFUNCTIONAL GREEN FLUORESCENT PROTELN AND USES THEREOF

[0001] This application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/347,833, filed October 24, 2001, and U.S. Provisional Patent Application Serial No. 60/371,691, filed April 10, 2002, which are hereby incorporated by reference in their entirety.

[0002] This subject matter of this application was made with support from the United States Government under National Science Foundation Grant No. BES97-12916 and the United States Department of Agriculture Grant No. 00-34135-9576. The U.S. Government may have certain rights.

FIELD OF THE INVENTION

[0003] The present invention relates to a multifunctional green fluorescent protein having a purification tag inserted within the β-barrel structure of the green fluorescent protein, and methods of using such a multifunctional green fluorescent protein.

BACKGROUND OF THE INVENTION

[0004] The green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria is one of the most widely used reporter proteins (Tsien, "The Green Fluorescent Protein," Annual Reviews in Biochemistry 67:509-544 (1998)). The utilization of GFP is superior to other reporter proteins, because direct visualization of gene expression and localization can be monitored without the need for invasive techniques and its detection requires no substrate or cofactors (Chalfie et al., "Green Fluorescent Protein as a Marker for Gene Expression," Science 263(5148):802-805 (1994); Heim et al., "Wavelength Mutations and Posttranslational Autooxidation of Green Fluorescent Protein," Proc. Natl. Acad. Sci. USA 91 :12501-12504 (1994)). GFP can also function as a protein tag, because it tolerates both N- and C-terminal fusions to a variety of proteins and can be expressed in a broad range of host organisms (Cubitt et al., "Understanding, Improving and Using Green Fluorescent Proteins," Trends in Biochemical Sciences 20:448-455 (1995)). [0005] The original GFP cloned from the jellyfish had a number of limitations, including low brightness, photoisomerization (Cubitt et al., "Understanding, Improving and Using Green Fluorescent Proteins," Trends in Biochemical Sciences 20:448-455 (1995)), and improper folding at 37°C (Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996)). This prompted considerable efforts to improve the spectral and folding properties of GFP (Tsien, "The Green Fluorescent Protein," Annual Reviews in Biochemistry 67:509-544 (1998)). One of the improved variants of GFP, designated GFP5, is thermostable and shows improved spectral properties (Siemering et al., "Mutations that

Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996)). GFP5 was developed by altering codon usage to disrupt a cryptic plant intron and by incorporating the mutations N163A, I167T, and S175G to increase thermotolerance and improve spectral properties (Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996)). This led to a GFP with dual excitation peaks at 395 nm and 473 ran of nearly equal amplitude. As a result, blue light could be used for excitation, unlike the wild-type (wt) GFP that needs to be excited with UN light. This alleviated the problem of photobleaching resulting from UN excitation and the harmful effects of UN irradiation. GFP5 expressed in bacterial cells that were grown at 37°C was shown to fluoresce 39-fold brighter than those expressing wt-GFP when excited at 395 nm and 111-fold brighter on excitation at 473 nm (Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996)). Mammalian Cos-7 cells grown at 37°C and expressing GFP5 were, on average, 20-fold brighter than those expressing wt-GFP when excited by blue light (Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996)). In order to express GFP5 in Arabidopsis, its codon usage was modified to eliminate plant intron recognition sequences. Expression level of GFP5 in plants was further enhanced by targeting GFP to the endoplasmic reticulum (ER) via addition of an Ν-terminal signal peptide and retaining GFP on the ER network via a C-terminal fusion with an ER-retention HDEL (His-Asp-Glu-Leu) signal (Haseloff et al., "Removal of a Cryptic Intron and Subcelmlar Localization of Green Fluorescent Protein are Required to Mark Transgenic Arabidopsis Plants Brightly," Proc. Natl. Acad. Sci. USA 94:2122-2127 (1997)). These reports demonstrate that GFP5 is a highly versatile variant that can be expressed in a variety of host organisms, with desirable spectral and folding properties.

[0006] Another widely adopted GFP mutation that improves its spectral properties involves replacement of serine in position 65 with threonine (S65T). This mutation results in GFP which fluoresces 6-fold more intensely when excited at 490 nm and the post-translational oxidation of the chromophore was 4-fold faster than wt-GFP (Heim et al., "Improved Green Fluorescence," Nature 373:663-664 (1995)). Moreover, Cos-7 cells expressing GFP5(S65T) fluoresced 1.65 times higher than cells expressing GFP5 grown at 37 °C (Siemering et al.,

"Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996). Like GFP5, S65T-type GFP can be excited by blue light, making it for on-line monitoring applications that require long exposures to strong light. S65T-type GFP has been highly expressed in transgenic Arabidopsis plants and non-disruptive fluorescence measurements were made in whole plants with blue light excitation (Niwa et al., "Non-Invasive Quantitative Detection and Applications of S65T-Type Green Fluorescent Protein in Living Plants," The Plant Journal 18(4 :455-463 (1999)).

[0007] Besides having improved spectral and folding properties, when using GFP as a fusion tag, it is desirable to introduce additional functionalities into GFP to expand its utility beyond merely a reporter. One such desirable function would be a high affinity for specific ligands to allow affinity purification. While affinity separations via GFP antibodies is feasible, scale-up of such an operation is costly. As an alternative approach, short peptide tags, such as a poly- histidine tag, have been used at the N- or C-terminus of a recombinant protein to aid in purification of recombinant proteins. For example, the glycosylated form of human fibroblast activation protein (FAP) was produced in insect cells and purified to near homogeneity using a hexa-histidine tag and immobilized metal affinity chromatography (IMAC) (Sun et al., "Expression, Purification, and Kinetic Characterization of Full-Length Human Fibroblast Activation Protein," Protein Expr. Purif. 24(2):274-281 (2002)). In another study, nearly pure human atrial natriuretic peptide (ANP) was produced and purified by metal affinity chromatography using an N-terminal His tag (Wilkinson et al., "Purification by Immobilized Metal Affinity Chromatography of Human Atrial Natriuretic Peptide Expressed in a Novel Thioredoxin Fusion Protein," Biotechnol. Prog. May- June 11(3):265-269 (1995)).

[0008] However, utility of peptide tags at termini of GFP fusion proteins has certain limitations. For example, it is often desirable to have an N-terminal ER-targeting signal peptide and a C-terminal ER-retention signal in a recombinant protein, as these signals generally enhance accumulation and stabilization of recombinant proteins. Such use of targeting/retention signals has been shown to be effective in enhancing recombinant protein accumulation in a wide range of host organisms including yeast, plant, animal, and insect cells. For example, a 5.4-fold increase in mouse single-chain antibody (ScFV) was seen in tobacco cell suspension culture when fused with a signal peptide of the tobacco pathogenesis related protein (PRla) at the N-terminus, a leader sequence at the N-terminus of the signal peptide, and a C-terminal KDEL, as compared to antibody not having the KDEL (Xu et al., "Combined Use of Regulatory Elements Within the cDNA to Increase the Production of a Soluble Mouse Single-Chain Antibody, scFv, from Tobacco Cell Suspension Cultures," Protein Expr. Purif. 24(3):384-394 (2002)). Moreover, when the ubiA gene from E. coli was expressed in tobacco with a C- terminal HDEL retention signal and a N-terminal ER-specific signal peptide, the activity of the expressed protein increased by about 116 times (Boehm et al., "Active Expression of the ubiA Gene from E coli in Tobacco: Influence of Plant ER-Specific Signal Peptides on the Expression of a Membrane-Bound Phenyltransferase in Plant Cells," Transgenic Res. Dec. 9(6):477-486 (2000)). [0009] Another limitation with N- or C- terminal peptide tags relates to interference with function. In particular, incorporation of a poly-histidine tag between the N-terminal signal peptide and the N-terminus of a GFP-fusion protein may interfere with the targeting function of the N-terminal signal peptide, due to the highly charged nature of the histidine tag. Likewise, putting a poly-histidine tag at the C-terminal may hamper proper functioning of the C-terminal ER- retention signal. As an alternative, the poly-histidine tag may be positioned in between the protein of interest and GFP. This has been demonstrated by the expression of GFP-XylE fusion protein in Sf9 insect cells with an in-frame 6xHis tag between the GFP and XylE cDNA (Wu et al., "Novel Freen Fluorescent Protein (GFP) Baculovirus Expression Vectors," Gene 190:157-162 (1997)). However, in this configuration, the probability that the 6xHis tag will be exposed for binding to an immobilized metal varies depending on the structure of the protein of interest and the overall conformation of the fusion protein. It is highly plausible that the folding pattern of the two proteins on either side of the 6xHis tag could render the tag inaccessible to the immobilized metal on an affinity column.

[0010] The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

[0011] The present invention relates to a multifunctional green fluorescent protein including a green fluorescent protein having a purification tag inserted within a β-barrel structure of the green fluorescent protein, where the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein.

[0012] The present invention also relates to a fusion protein including a green fluorescent protein having a purification tag inserted within a β-barrel structure of the green fluorescent protein, where the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, and at least one protein or polypeptide operably linked to the green fluorescent protein. [0013] The present invention also relates to nucleic acids, host cells, and an expression system containing the multifunctional green fluorescent protein of the present invention. [0014] Another aspect of the present invention relates to a method for purifying a protein or polypeptide. This method involves providing a fusion protein including a green fluorescent protein having a purification tag inserted within a β-barrel structure of the green fluorescent protein operably linked to at least one protein or polypeptide, contacting the fusion protein with a substrate having a binding affinity for the purification tag under conditions effective to bind the purification tag to the substrate, and isolating the at least one protein or polypeptide from the bound fusion protein. [0015] The present invention also relates to a second method for purifying a protein or polypeptide. This method involves providing a fusion protein including a green fluorescent protein having a purification tag inserted within a β- barrel structure of the green fluorescent protein operably linked to at least one protein or polypeptide, wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, and isolating the at least one protein or polypeptide from the fusion protein based on the change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein. [0016] Another aspect of the present invention relates to a method for identifying expression of protein or polypeptide in biological material. This involves providing a nucleic acid construct including a first nucleic acid molecule encoding a green fluorescent protein having a purification tag, wherein the purification tag is inserted into a β-barrel structure of the green fluorescent protein and wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, operably linked to at least one second nucleic acid molecule encoding at least one protein or polypeptide. The first and the at least one second nucleic acid molecules are operably linked to 5' and 3' regulatory nucleic acid molecules that allow expression of the first and at least one second nucleic acid molecules, and expression of the first and at least one second nucleic acid molecules produces a green fluorescent protein-at least one protein or polypeptide fusion protein. This method also involves introducing the nucleic acid construct to a biological material under conditions effective to allow expression of the fusion protein in the biological material, and detennining fluorescence of the green fluorescent protein in the biological material to identify expression of at least one protein or polypeptide in the biological material. [0017] Another aspect of the present invention relates to a method for identifying a target molecule. This involves providing a support having a plurality of green fluorescent protein molecules immobilized at particular sites on the support, where each green fluorescent protein molecule contains a different affinity binding peptide tag inserted within a β-barrel structure of the green fluorescent protein, providing a sample potentially containing one or more target molecules, where each target molecule has a binding affinity for a specific affinity binding peptide tag of the green fluorescent protein molecules, treating the sample with a fluorophore having a different excitation and emission peak than that of green fluorescent protein under conditions effective to label the one or more target molecules with the fluorophore, if present in the sample, contacting the support with the sample under conditions effective to bind the one or more target molecules to the specific affinity binding peptide tags, and detecting a change in fluorescence of at least one of the green fluorescent protein molecules caused by binding of the one or more target molecules to the specific affinity binding peptide tags to identify the one or more target molecules.

[0018] Yet another aspect of the present invention relates to a microarray including a plurality of green fluorescent protein molecules immobilized at particular sites on a support, wherein each green fluorescent protein molecule comprises a different affinity binding peptide tag inserted within a β-barrel structure of the green fluorescent protein.

[0019] The GFP molecule of the present invention allows maximum flexibility for protein/polypeptide fusion, because both N- and C-terminal ends are available for linking to a protein/polypeptide of interest. In conjunction with appropriate targeting/retention signals, this GFP tag can improve fusion protein accumulation and stability and, at the same time, perform the multiple functions of protein/polypeptide monitoring, identification, and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a 3-D diagram of the structure of the green fluorescent protein (GFP) showing the location of amino acids 172Glu and 173 Asp in an exposed solvent loop of GFP, where the hexa-histidine (6xHis) tag was inserted. The N- and C- terminal ends of GFP are also shown.

[0021] Figure 2 is the nucleic acid and corresponding amino acid sequence for the green fluorescent protein variant GFP 172. The 6 histidines are shown in bold. Ncol and Sacl sites at the Ν- and C-terminus are underlined. The S65T mutation in the chromophore (TYG) is shown. Since an Ncol site was introduced at the Ν-terminus, an internal Ncol site was removed by a single base change without changing the codon (boxed).

[0022] Figure 3 shows the effect of the S65T variant on GFP5 fluorescence intensity readings for 5 μg/ml of GFP in GFP extraction buffer.

Intensities were measured with excitation set at 467 nm and emission set at 505 nm for GFP 172 and 490 nm excitation and 505 nm emission for GFPS65T172.

[0023] Figures 4A-B are SDS-PAGE gels comparing the solubilities of the

GFP variants (with the S65T mutation) during expression in bacterial cells at 37°C, shown in Figure 4A, and 28°C, shown in Figure 4B. Samples equivalent to 100 μl of cell culture were loaded on a 12% polyacrylamide gel. I = Insoluble fraction; S = Soluble fraction.

[0024] Figure 5 is a graph showing a comparison of fluorescence intensity readings for 5 μg/ml of GFP in GFP extraction buffer produced at different temperatures. Readings were taken by setting the excitation at 490 nm and emission at 512 nm. Each intensity reading is an average of 4 replicates with each replicate reading an average of intensity measure at time intervals of 10 seconds, 30 seconds, and 60 seconds after placing the cuvette in the F-2500 fluorescent spectrophotometer.

[0025] Figure 6 shows excitation and emission spectra for GFP variants

GFPHis (top panel) and GFP 172 (bottom panel) at 37°C. Protein concentrations were 5 μg/ml in GFP extraction buffer (pH 8.0) and samples were allowed to stabilize for 1 minute after mixing before the scan was performed. All spectra have been normalized to a maximum value of 1.0.

[0026] Figure 7 shows excitation and emission spectra for the GFP variants, GFPHis, GFP172, and GFP157, (top to bottom panels) produced at 28°C. Protein concentrations were 5 μg/ml in GFP extraction buffer (pH 8.0) and samples were allowed to stabilize for 1 minute after mixing before the scan was performed. All data points of the spectra have been normalized based on the peak height of the GFPHis variant.

[0027] Figure 8 is an SDS-PAGE gel showing protein samples expressed in E. coli and purified by immobilized metal affinity chromatography (IMAC). Proteins were quantified by BCA assay using recombinant GFP (Clontech, Palo Alto, CA) as standard. A total of 2 μg protein was loaded on 12% polyacrylamide gel and stained with Coomassie Brilliant Blue. M and Std denote the protein ladder (Invitrogen, Carlsbad, CA), and a GFP standard without 6xHis tag (Clontech, Palo alto, CA), respectively.

[0028] Figure 9 is a graph showing GFP recovery experiments (in duplicate). A total of 20 μg of GFP172, GFP157, or GFPHis was spiked into extraction buffer and the resulting fluorescent intensity measured. Protein was recovered using IMAC. Protein was eluted in 1 ml fractions to a total of 3 ml. Fluorescence of each eluted fraction was measured by setting excitation at 490 nm and emission at 512 nm. GFP fluorescence was highly linear in the 0.5- 20 μg/ml in extraction buffer and there was hardly any change in linearity when fluorescence was measured in elution buffer. The fluorescence of eluted sample gives a measure of GFP recovered by 6xHis tag metal affinity chromatography. [0029] Figure 10 is a graph showing relative fluorescence as a function of pH for GFP 172, GFP 157, and GFPHis. Intensity readings were made for 5μg/ml of GFP variants, as noted, (produced at 28°C) at 10 seconds, 30 seconds, and 60 seconds and averaged. The averaged values were normalized to a maximum value of 1.0. The data was curve fitted as described in Example 15, below. [0030] Figure 11 is a gel showing MBP-GFP172 purified from E. coli lysate. M: Benchmark Protein Ladder (Invitrogen , Carlsbad, CA), Lane 1 : E. coli lysate equivalent to lOOμl cell culture; Lane 2: fusion protein purified by IMAC (3-5μg).

[0031] Figure 12 is an SDS-PAGE gel demonstrating GFP-fusion protein fluorescence. Samples were loaded without boiling on 10% SDS-PAGE gels. Lane 1: 10 μg of MBP-GFP172; Lane 2: 4 μg of GFP172.

[0032] Figure 13 is a Coomassie stained gel showing the purification of the MBP-GFP172-fusion protein from tobacco extract using IMAC. Lane 1: lOOμg of soluble proteins from tobacco extract; Lane 2: 3μg of eluted fraction having the highest fluorescence; Lane 3: 2μg of fusion protein purified from E. coli; Lane 4: 3.0 μg of fusion protein purified from E. coli.

[0033] Figure 14 is a standard curve of MBP-GFP172 fusion protein in elution buffer (50mM NaH2PO4, 300mM NaCl, 150 mM imidazole pH 7.0). Fusion protein purified from E. coli was made up in elution buffer at different concentrations and fluorescence measured with excitation set at 490 nm and emission set at 512 nm.

[0034] Figure 15 is an SDS-PAGE gel showing GFP detection by fluorescence. Lane 1 : 2μg fusion protein purified from E. coli; Lane 2: 2μg GFP 172; Lane 3: 2μg of MBP-GFP172 purified from tobacco extract (with no phenylmethyl sulfonyl fluoride (PMSF); Lane 4: 2μg of MBP-GFP172 purified from tobacco extract (with ImM PMSF); Lane 5: 3μg of MBP-GFP172 purified from tobacco extract (with ImM PMSF); Lane 6: lOOng of MBP-GFP172; Lane 7: lOOng of GFP172; Lane 8: 500ng of GFP172; Lane 9: lμg of GFP172.

[0035] Figure 16 is a gel showing Factor Xa cleavage of a MBP-GFP fusion protein at different concentrations of Factor Xa. Each lane represents 5μls of a 1 μg/μl sample taken at different time intervals using 200ng Factor Xa.

M=Benchmark protein ladder; Lane 1: uncut fusion (0 hours); Lane 2: 6 hours; Lane 3: 24 hours; Lane 4: 40 hours; Lane 5: complete cleavage at 6 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention relates to a multifunctional green fluorescent protein including a green fluorescent protein having a purification tag inserted within a β-barrel structure of the green fluorescent protein, where the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein. [0037] As shown in Figure 1, GFP is a compact molecule having a β- barrel (also called a "β-can") structure formed by 11-antiparallel beta strands to form a compact cylinder with some of the connecting loops exposed to solvent and the chromophore buried inside the cylinder (Ormo et al., "Crystal Structure of the Aequorea Green Fluorescent Protein," Science 273:1392-1395 (1996); Yang et al., "The Molecular Structure of Green Fluorescent Protein," Nature

Biotechnology 14:1246-1251 (1996), which are hereby incorporated by reference in their entirety). The present invention relates to introducing a purification tag within the polypeptide chain of the GFP molecule (i.e., not at the termini of the GFP molecule), preferably, within a surface loop (i.e., solvent exposed loop) of GFP, thereby producing a tagged GFP which overcomes the restrictions posed by current GFP-fusion proteins. In particular, the multifunctional GFP variant of the present invention can be used not only as a reporter tag, but also a purification tag, independent of the conformation of the fusion partner or the requirement for additional fusions on both termini of the GFP-fusion protein thereto (i.e., with a broad range of protein (peptide fusion strategies).

[0038] Suitable purification tags for insertion within the GFP molecule of the present invention include any peptide that introduces specific molecular recognition properties (e.g., peptide affinity tags) or other unique properties that allow selective separation and recovery for a protein or polypeptide of interest. This includes proteins or polypeptides that bind other proteins, for example, ligand-binding proteins, enzymes, antibodies, and selective epitopes thereof. However, the properties of the purification tag need not be limited to protein binding. Other suitable purification tags for the present invention include peptides with other properties of specificity, including, without limitation, metal-binding peptides (e.g., poly-histidine, which can be purified by IMAC or precipitation), carbohydrate-binding domains (e.g., starch binding domain (SBD), cellulose binding domain (CBD), maltose binding domain (MBD)), biotin-binding domains, antigenic epitopes, and nucleic acid-binding peptides. Also suitable as purification tags in the present invention are amino acids having other unique properties that allow highly selective separation and/or recovery. For example, the purification tag may be an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the GFP. This includes, without limitation, charged amino acids, such as poly(Arg) (e.g., 5-15 aa), poly(Asp) (e.g., 5-16 aa), and poly-histidine, which can be used in charge-based recovery schemes, for example, separation based on ion exchange, precipitation using polyelectrolytes having opposite charges, aqueous two-phase partitioning, and reversed micellar extraction (Glatz et al., "Genetic Engineering to Enhance the Selectivity of Protein Separations," Applied Biochemistry and Biotechnology 54: 173-191 (1995); Berggren et al., "Peptide Fusion Tags with Tryptophan and Charged Residues for Control of Protein Partitioning in PEG- Potassium Phosphate Aqueous Two-Phase Systems," Bioseparation 9(2):69-80 (2000); Berggren et al., "Genetic Engineering of Protein-Peptide Fusions for Control of Protein Partitioning in Thermoseparating Aqueous Two-Phase Systems," Biotechnology and Bioengineering,, 62(2):135-144 (1999); Fan et al, "Contribution of Protein Charge Partitioning in Aqueous Two-Phase Systems," Biotechnology and Bioengineering, 59(4):461-470 (1998); Luther et al., "Genetically Engineered Charge Modifications to Enhance Protein Separation in Aqueous Two-Phase Systems: Charge Directed Partitioning," Biotechnology and Bioengineering 46(1): 62-68 (1995); Forney et al., "Extraction of Charged Fusion Proteins in Reversed Micelles: Comparison Between Different Surfactant Systems," Biotechnology Progress, ll(3):260-264 (1995); Forney et al., "Reversed Micellar Extraction of Charged Fusion Proteins," Biotechnology Progress 10(5):499-502 (1994), which are hereby incorporated by reference in their entirety). In charge-directed partitioning, for example, a protein tagged with charged amino acids is preferentially directed to one phase in two-phase aqueous separation. This results from both electrostatic as well as non-electrostatic effects (Berggren et al., "Genetic Engineering of Protein-Peptide Fusions for Control of Protein Partitioning in Thermoseparating Aqueous Two-Phase Systems," Biotechnology and Bioengineering, 62(2):135-144 (1999); Fan et al, "Contribution of Protein Charge Partitioning in Aqueous Two-Phase Systems," Biotechnology and Bioengineering, 59(4):461-470 (1998), which are hereby incorporated by reference in their entirety). Also suitable as a purification tag are elastin-like polypeptides (ELPs). Elastin-like polypeptides are peptides having oligomeric repeats of the pentapeptide sequence Val-Pro-Gly-Xaa-Gly (SEQ ID NO: 1), where Xaa can be any amino acid except Pro. The ELPs are thermally responsive polypetides that undergo reversible aggregation above a threshold temperature. In particular, ELPs give rise to a fluid-solid phase transition above a specific transition temperature, therefore, the use of ELPs as a GFP tag allows for separations by thermally-induced aggregation and precipitation. According to Meyer et al., "Protein Purification By Fusion With An Environmentally Responsive Elastin-like polypeptide: Effect of Polypeptide Length on The Purification of Thioredoxin," Biotechnology Progress 17:720-728 (2001), an ELP as small as 9 kDa still allows efficient purification by thermally induced precipitation. Also suitable as tags are poly(Phe) (e.g., 11 aa),( for use with

Phenyl-Superose for separation/recovery), and poly(Cys) (e.g., 4 aa) (for use with Thiopropyl-Sepharose for separation/recovery). In principal, a vast variety of amino acid sequences may be selected from combinatorial peptide libraries against a wide range of ligands as targets, including non-protein ligands (e.g., nucleic acid ligands or aptamers). Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acids, proteins, small organic compounds, and even entire organisms. These novel molecules have many potential uses in medicine and technology. It is also possible to select amino acid sequences that can be captured by molecularly imprinted polymers. [0039] In one embodiment of the present invention, the purification tag has from about 4 to about 20 amino acid residues. In a preferred embodiment, the purification tag is a polyhistidine (e.g., hexa-histidine) tag. The use of a polyhistidine tag allows for purification of a target protein under native conditions using IMAC, based on the high affinity of the polyhistidine tag towards metal ions. In addition, the use of a polyhistidine tag may result in a multifunctional GFP that exhibits high tolerance to low pH. This is particularly relevant when recombinant-therapeutic proteins, including GFP, are to be expressed in plants, as the media prepared for callus and plant cells is typically at pH 5.5. While some tags or conditions of use may either enhance or decrease the intensity of the tagged GFP of the present invention, the alteration of the spectral properties of GFP is not required in the present invention. A His-tag may also be used for purification procedures that are not based on binding properties. For example, a poly-histidine tag may be used in charge-based recovery schemes, as described herein. [0040] In another embodiment of the present invention, more than one purification tag is inserted within a single GFP molecule. The use of more than one tag provides increased functionality for the GFP by increasing the variety of schemes available for purification, separation, recovery, and identification of polypeptides and proteins of interest. [0041] Suitable locations for insertion of the purification tag include surface loops of the GFP, as well as other areas of the β-barrel structure of a GFP molecule (see, e.g., Yang et al., Nature Biotech. 14:1246-1251 (1996), which is hereby incorporated by reference in its entirety) which do not compromise chromophore function. Examples of suitable sites within the β-barrel region of

GFP include, without limitation, sites Glnl57-Lysl58, Glul72-Aspl73, and Leul94-Leul95 (Abedi et. al., "Green Fluorescent Protein as a Scaffold for Intracellular Presentation of Peptides," Nucleic Acids Research 26: 623-630 (1998), which is hereby incorporated by reference in its entirety). In one report, a GFP based biosensor was designed by inserting a binding domain between amino acids 172 and 173 and an increase in fluorescence was noted when ligand was bound (Doi and Yanagawa, "Design of Generic Biosensors Based on Green Fluorescent Proteins with Allosteric Sites by Directed Evolution," FEBS Lett. 453: 305-307 (1999), which is hereby incorporated by reference in its entirety). By inserting the purification tag into a surface loop of the GFP, the fluorescent activity of GFP remains, while the peptide affinity tag is suitably positioned for affinity purification.

[0042] The multifunctional GFP of present invention is prepared by manipulating a nucleic acid molecule encoding GFP, either the wild-type (wt- GFP) or a variant of wt-GFP, as desired, to insert a protein or polypeptide tag within the β-barrel region of the 11 -stranded β-barrel GFP structure, for example, into a surface loop. Generally, this involves obtaining the nucleic acid molecule for the GFP of interest, and modifying the sequence of the molecule using molecular biology techniques, including PCR amplification and site-directed mutagenesis, to insert the sequence for a purification tag of choice into a desired site between two amino acids in an of a β-barrel region within the GFP molecule.

Such techniques are known in the art and described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety. The GFP molecule can be a wild-type GFP or a variant, including but not limited to, the mGFP5 variant, the GFP5-S65T variant, a cyan fluorescent protein variant, a yellow fluorescent protein variant, a red fluorescent protein variant, and a blue fluorescent protein variant. Such variants are known and described in the art (see, e.g., Tsien. R.Y., " The Green Fluorescent Protein," Annual Reviews in Biochemistry 67:509-544 (1998); Cormack et al., " FACS-Optimized Mutants of the Green Fluorescent Protein (GFP)," Gene 173:33-38 (1996); Cubitt et al.; "Understanding, Improving and Using Green Fluorescent Proteins," TIBS 20:448-455 (1995), which are hereby incorporated by reference in their entirety). The method of making such a multifunctional GFP molecule is described in greater detail in Example 1, below. [0043] Another aspect of the present invention relates to a fusion protein including the multifunctional green fluorescent protein of the present invention, wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, and at least one protein or polypeptide of interest operably linked to the green fluorescent protein. The protein or polypeptide of interest can be any protein or polypeptide that one desires to produce as a recombinant protein or polypeptide, whether prokaryotic or eukaryotic in nature. [0044] The present invention also relates to a nucleic acid construct encoding the multifunctional green fluorescent protein of the present invention. In particular, the present invention relates to chimeras of GFP, as described above, wherein the green fluorescent protein has been engineered to contain a purification tag within the β-barrel structure of the GFP, and is operably linked to at least one protein or polypeptide of interest. To create the chimera, a nucleic acid construct is made in which a first nucleic acid encoding the GFP having a purification tag, and at least one second nucleic acid molecule encoding a protein or polypeptide of interest, are operably linked to each other and to 5' and 3 ' regulatory nucleic acid molecules that allow expression of the first and second nucleic acid molecules. Expression of the first and at least one second nucleic acid molecules produces a green fluorescent protein-at least one protein or polypeptide fusion protein. Constructs can also be made with the first nucleic acid encoding the at least one protein or polypeptide of interest and the second encoding GFP. In this conformation, the GFP can also be used as a folding reporter, because the folding pattern of the upstream protein correlates with fluorescence (Waldo et al., "Rapid Protein-Folding Assay Using Green Fluorescent Protein," Nature Biotech. 17:691-695 (1999), which is hereby incorporated by reference in its entirety).

[0045] Nucleic acid constructs of the present invention are prepared by incorporating the nucleic acid molecules encoding the proteins/polypeptides of choice into a suitable vector and subsequent propagation of the vector in suitable host cells. This involves the practice of conventional recombinant DNA technology. Generally, this involves first inserting the nucleic acid molecule into an expression system to which the nucleic acid molecule is heterologous (i.e., not normally present). When suitable, the heterologous nucleic acid molecule is inserted into the expression system which includes the necessary elements for the transcription and translation of the inserted protein coding sequences, producing what is termed herein a fusion protein.

[0046] The nucleic acid molecules of the present invention may be inserted into any of the many available expression vectors using reagents that are well known in the art. Suitable vectors include, but are not limited to, the following viral vectors: lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, CA, which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pET series (see F.W. Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof. In preparing the nucleic acid constructs of the present invention, the various nucleic acid molecules of the present invention may be inserted or substituted into a bacterial plasmid- vector. Any convenient plasmid may be employed, which will be characterized by having a bacterial replication system, a marker which allows for selection in a bacterium and generally one or more unique, conveniently located restriction sites. Numerous plasmids, referred to as transformation vectors, are available for transformation. The selection of a vector will depend on the preferred transformation technique and target cells for fransfection. The selected and prepared nucleic acid molecules of the present invention are cloned into the vector using standard cloning procedures in the art, such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety.

[0047] U.S. Patent No. 4,237,224 issued to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture. [0048] Certain "control elements" or "regulatory sequences" are also incorporated into the plasmid- vector constructs of the present invention. These include non-transcribed regions of the vector and 5' and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5' promoter elements may be used.

[0049] A constitutive promoter is a promoter that directs constant expression of a gene in a cell. Examples of some constitutive promoters that are widely used for inducing expression of transgenes include the nopoline synthase ("NOS") gene promoter, from Agrobacterium tumefaciens (U.S. Patent No. 5,034,322 issued to Rogers et al., which is hereby incorporated by reference in its entirety), the cauliflower mosaic virus ("CaMV") 35S and 19S promoters (U.S. Patent No. 5,352,605 issued to Fraley et al., which is hereby incorporated by reference in its entirety), those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Patent No. 6,002,068 issued to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter ("ubi"), which is the promoter of a gene product known to accumulate in many cell types. Examples of constitutive promoters for use in mammalian cells include the RSN promoter derived from Rous sarcoma virus, the CMN promoter derived from cytomegalo virus, and the EFlα promoter derived from the cellular elongation factor lα gene. [0050] Also suitable as a promoter in the plasmids of the present invention is a promoter that allows for external control over the regulation of gene expression. One way to regulate the amount and the timing of gene expression is to use an inducible promoter. Unlike a constitutive promoter, an inducible promoter is not always optimally active. An inducible promoter is capable of directly or indirectly activating transcription of one or more DΝA sequences or genes in response to an inducer. Some inducible promoters are activated by physical means such as the heat shock promoter ("Hsp"). Others are activated by a chemical, for example, IPTG or tetracycline ("Tet on" system). Other examples of inducible promoters include the metallothionine promoter, which is activated by heavy metal ions, and hormone-responsive promoters, which are activated by treatment of certain hormones. In the absence of an inducer, the nucleic acid sequences or genes under the control of the inducible promoter will not be transcribed or will only be minimally transcribed. When any plasmids of the present invention contain an inducible promoter, the method of the present invention further includes the step of adding an appropriate inducing agent to the cell culture when activation of the promoter is desired. [0051] Plasmids of the present invention also include operable 3 ' regulatory elements, selected from among those elements which are capable of providing correct transcriptional termination and proper polyadenylation of mRNA for expression in the host cell of choice, operably linked to a DNA molecule which encodes a protein of choice. Exemplary 3' regulatory elements include, without limitation, the nopaline synthase ("nos") 3' regulatory region (Fraley, et al., "Expression of Bacterial Genes in Plant Cells," Proc. Nat'l Acad. Sci. USA 80(15):4803-4807 (1983), which is hereby incorporated by reference in its entirety) and the cauliflower mosaic virus ("CaMV") 3' regulatory region (Odell, et al., "Identification of DNA Sequences Required for Activity of the Cauliflower Mosaic Virus 35S Promoter," Nature 313(6005):810-812 (1985), which is hereby incorporated by reference in its entirety). An example of a commonly-used 3' regulatory element for expression of genes of interest in mammalian cells is the SV40 polyadenylation signal derived from the SV40 virus. Virtually any 3' regulatory element known to be operable in the host cell of choice will suffice for proper expression of the nucleic acid molecule contained in the chimera of the present invention.

[0052] A vector of choice, a suitable promoter, nucleic acid molecules specific to each plasmid as described above, an appropriate 3' regulatory region, as well as other regulatory element(s) if appropriate, can be used to construct the nucleic acid constructs of this aspect of the present invention, using well known molecular cloning techniques in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1989), which are hereby incorporated by reference in their entirety. Once constructed, the plasmids of this aspect of the present invention can be amplified by propagation in a suitable host cell (e.g., inE. coli) and subsequently produced in a larger quantity. Methods of producing plasmids in desired quality and quantity are well known in the art. [0053] Once an expression plasmid construct of the present invention has been prepared in sufficient quality and quantity, it is ready to be incorporated into a suitable host cell to practice the method described in this aspect of the present invention. Basically, this is carried out by transforming or transfecting a host cell with a plasmid of the present invention, using standard procedures known in the art, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), which is hereby incorporated by reference in its entirety. Suitable host cells for the present invention include, without limitation, bacterial cells, yeast cells, plant cells, and mammalian cells, including human cells, as well as any other cell system that is suitable for producing a recombinant protein. Methods of transformation or fransfection may result in transient or stable expression of the genes of interest contained in the plasmids. Transient expression by the host cells of the present invention is sufficient for carrying out the present invention, although stable expression is also suitable. Stable expression may be more desirable in certain applications of the present invention. Methods of transforming and transfecting host cells are well known in the art. Examples of suitable methods for transfecting mammalian cells include, without limitation, calcium phosphate coprecipitation, electroporation, and lipofection. Following transformation or fransfection, the cells are then cultured in a suitable way as per the specific cell type, the desired expression (transient vs. stable), and the nature of the reporter whose activity is going to be assayed.

[0054] In the case when a stable expression of a nucleic acid molecule of interest is desired, stably transfected cells can be identified using a selection marker simultaneously introduced into the host cells along with the plasmid construct of the present invention. Usually, the selection marker is contained in the plasmid. Suitable selection markers include, without limitation, markers encoding for antibiotic resistance, such as the neomycin resistance gene and the hygromycin resistance gene (Southern and Berg, "Transformation of Mammalian Cells to Antibiotic Resistance With a Bacterial Gene Under the Control of the

SV40 Early Region Promoter," J Mol Appl Genet., 1(4):327-41 (1982); Bernard et al., "Construction of a Fusion Gene That Confers Resistance Against Hygromycin B to Mammalian Cells in Culture," Exp Cell Res. 158(l):237-43 (1985), which are hereby incorporated by reference in their entirety). [0055] The present invention also relates to a method for purifying a protein or polypeptide. This method involves providing a fusion protein including a green fluorescent protein having a purification tag inserted within a β-barrel structure of the green fluorescent protein operably linked to at least one protein or polypeptide, contacting the fusion protein with a substrate having a binding affinity for the purification tag under conditions effective to bind the purification tag to the substrate, and isolating the at least one protein or polypeptide from the bound fusion protein. As used herein, isolating the at least one protein or polypeptide from the fusion protein includes recovering the fusion protein from the substrate, and processing the recovered fusion protein to separate the at least one protein or polypeptide from the GFP molecule. Suitable GFP molecules, purification tags, protein or polypeptide molecules which are operably linked to the GFP molecule, and the method of making such a nucleic acid construct are as described above, including the choice of suitable vectors, 5' and 3' regulatory regions, other regulatory element(s) when appropriate, host cells, and method of making. After the fusion protein is made as described above, the method generally involves expressing the fusion protein, for example, in either a prokaryotic or eukaryotic cell system having a liquid medium suitable for sustaining cells capable of expressing the GFP-at least one protein or polypeptide fusion product, or a cell-free system having a buffer solution suitable for expressing the fusion product. The fusion protein in solution can be contacted with a substrate having a binding affinity for the purification tag and eluted from the substrate in discrete fractions. The fluorescence of the fractions can be monitored throughout the process, or can be determined at the end point of recovery, thereby identifying the one or more fractions containing the fusion protein. If desired, the identified fusion protein can be processed to separate the green fluorescent protein from the at least one protein or polypeptide, and the at least one protein or polypeptide is collected. The use of affinity substrates for binding to a protein is well-known in the art. The choice of substrate will be dependent upon the purification tag inserted in the GFP. For example, when the GFP purification tag is a poly-histidine, the fusion protein solution may be purified by immobilized metal affinity chromatography (IMAC) (described in greater detail in Example 4, below), or using charge-based techniques, as described herein. Suitable substrates include substrates for the purification tags described herein. Regardless of the purification tag-affinity substrate scheme chosen, the location of the fusion protein can be monitored throughout the purification process by determining where sample fluorescence is occurring. For example, in the embodiment in which column chromatography is used to purify the expressed fusion protein, fluorescence can be monitored as the fusion protein travels through the column, and/or in the fractions as they are eluted from the column. The fractions which fluoresce are associated with the desired protein or polypeptide and are collected. The desired protein or polypeptide is then separated from GFP to produce a purified protein or polypeptide. This generally involves a step in which the fusion proteins are cleaved chemically, for example, by treatment with the restriction protease Factor Xa, which recognizes the amino acid sequence Ile-Glu/Asp-Gly-Arg, and cleaves the peptide bond C-terminal of the arginine residue. (See Examples 10 and 18 for a more detailed description of the use of Factor Xa.) Also suitable as cleavage sites are those recognized by enterokinase, genenase I, and cyano bromide (for a review of suitable cleavage techniques see Glatz et al., "Genetic Engineering to Enhance the Selectivity of Protein Separations," Applied Biochemistry and Biotechnology 54: 173-191 (1995), which is hereby incorporated by reference in its entirety). The two proteins are then separated, and the desired recombinant protein is recovered. The desired separation sites are determined prior to the making of the fusion protein, and the desired cutting site is engineered into the expression vector into which the nucleic acid molecules for GFP and the at least one protein or polypeptide are incorporated using conventional molecular biology methods as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety. [0056] The present invention also relates to a second method for purifying a protein or polypeptide. This method involves providing a fusion protein including a green fluorescent protein having a purification tag inserted within a β- barrel structure of the green fluorescent protein operably linked to at least one protein or polypeptide, wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, and isolating the at least one protein or polypeptide from the fusion protein based on the change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein. In this aspect of the present invention, suitable purification tags are those described above that are capable of introducing a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, such as charged amino acids (e.g., poly(Arg) (e.g., 5-15 aa), poly(Asp) (e.g., 5-16 aa), and poly-histidine), which can be used in charge-based recovery schemes, for example, separation based on ion exchange, precipitation using polyelectrolytes having opposite charges, aqueous two-phase partitioning, and reversed micellar extraction (Glatz et al., "Genetic Engineering to Enhance the Selectivity of Protein Separations," Applied Biochemistry and Biotechnology 54: 173-191 (1995), which is hereby incorporated by reference in its entirety), elastin-like polypeptides (which allow for separations by thermally-induced aggregation and precipitation), poly(Phe) (e.g., 11 aa),( for use with Phenyl- Superose for separation/recovery), and poly(Cys) (e.g., 4 aa) (for use with Thiopropyl-Sepharose for separation/recovery). Isolating, as used in this aspect of the present invention, will be carried out in accordance with the purification tag selected, as described above. A change in environmental conditions may or may not be required to trigger the change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein. [0057] Another aspect of the present invention relates to a method for identifying expression of protein or polypeptide in biological material. This involves providing a nucleic acid construct including a first nucleic acid molecule encoding a green fluorescent protein having a purification tag, wherein the purification tag is inserted into a β-barrel structure of the green fluorescent protein and wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, operably linked to at least one second nucleic acid molecule encoding at least one protein or polypeptide. The first and the at least one second nucleic acid molecules are operably linked to 5' and 3' regulatory nucleic acid molecules that allow expression of the first and at least one second nucleic acid molecules, and expression of the first and at least one second nucleic acid molecules produces a green fluorescent protein-at least one protein or polypeptide fusion protein. This method also involves introducing the nucleic acid construct to a biological material under conditions effective to allow expression of the fusion protein in the biological material, and determining fluorescence of the green fluorescent protein in the biological material to identify expression of at least one of the protein or polypeptide in the biological material. [0058] The nucleic acid construct of the this aspect of the present invention is a GFP chimera as described above. Suitable GFP molecules, protein or polypeptide fusion proteins, purification tags, and method of making such a nucleic acid construct are carried out as described above, including the choice of suitable vectors, 5' and 3' regulatory regions, other regulatory element(s) when appropriate, host cells, as well as necessary methodology available in the art. Once the nucleic acid construct is prepared in a suitable expression system, the construct is introduced into a biological material. The biological material may be a cell, tissue, bone, or body fluid. The source of the biological material can be bacteria, yeast, plant, or mammal. The vectors, host cells, and method of introduction are selected in accordance with the source and type of biological material chosen. Methods of introduction include, without limitation, those described above for the nucleic acid construct of the present invention, and are selected in accord with the biological material. As noted above, expression levels using a GFP having a purification tag inserted in the exposed loop allows both the N-terminal and C-terminal ends of GFP to be available for the addition of useful molecules in the fusion protein construct. For example, the expression level of GFP5 in plants was further enhanced by targeting GFP to the endoplasmic reticulum (ER) via addition of an N-terminal signal peptide and retaining GFP on the ER network via a C-terminal fusion with an ER-retention HDEL signal (Haseloff et al., "Removal of a Cryptic Intron and Subcellular Localization of Green Fluorescent Protein are Required to Mark Transgenic Arabidopsis Plants Brightly," Proc. Natl. Acad. Sci. USA 94:2122-2127 (1997), which is hereby incorporated by reference in its entirety). This concept can be utilized to enhance the expression of the fusion protein by tailoring the nucleic acid construct specifically to a biological material or location of interest in the material. Once the nucleic acid construct is introduced into the material of choice, the occurrence of fluorescence is determined within the sample, allowing for the identification/localization of expression of the protein or polypeptide of interest. [0059] A further aspect of the present invention relates to a method for identifying a target molecule. This method involves providing a support having a plurality of green fluorescent protein molecules immobilized at particular sites on the support, wherein each green fluorescent protein molecule includes a different affinity binding peptide tag inserted within a β-barrel structure of the green fluorescent protein, providing a sample potentially containing one or more target molecules, wherein each target molecule has a binding affinity for a specific affinity binding peptide tag, treating the sample with a fluorophore, wherein the fluorophore has a different excitation and emission peak than that of green fluorescent protein under conditions effective to label the one or more target molecules with the fluorophore, if present in the sample, contacting the support with the sample under conditions effective to bind the one or more target molecules to the specific affinity binding peptide tags, and detecting a change in fluorescence of at least one of the green fluorescent protein molecules caused by binding of the one or more target molecules to the specific affinity binding peptide tags to identify the one or more target molecules.

[0060] Suitable affinity binding peptides include the purification tags described above. Target molecules in this method of the present invention may be virtually any type of molecule, including, without limitation, proteins, polypeptides, and nucleic acid molecules, either eukaryotic or prokaryotic in nature. An exemplary fluorophore of this aspect of the present invention is rhodamine, however, other suitable fluorophores known to those in the art may be used. In particular, suitable fluorophores include fluorophores which do not have the same excitation and emission properties as GFP, and thereby allow fluorescence resonance energy transfer (FRET) with the green fluorescent protein when both are present in close proximity. In particular, after the immobilized GFP is contacted with the sample potentially containing fluorophore labeled target molecules, the support is exposed to a wavelength suitable to excite the GFP fluorophore, which, in turn, excites the second, different fluorophore of the target molecules. If the target molecule has binding affinity for the affinity peptide tag of an immobilized GFP molecule, binding will bring the GFP and target molecules into close proximity, thereby allowing FRET to occur. Detection of FRET identifies a target molecule. FRET is described in more detail in Mitra et al., "Fluorescence Resonance Energy Transfer Between Blue-Emitting and Red- Shifted Excitation Derivatives of the Green Fluorescent Protein," Gene 173:13-17 (1996), which is hereby incorporated by reference in its entirety. Briefly, FRET is a process in which an excited fluorophore (the donor) transfers its excited state energy to a light absorbing molecule (the acceptor). In one embodiment, the GFP molecule is excited first, and as "donor," transfers its energy to the target "acceptor" molecule, creating a detectable change in wavelength emitted. [0061] The GFP molecules are immobilized on a support, which may include, without limitation, beads, plastic supports, silicon supports, a protein chip, a multiwell device, glass supports (e.g., a glass slide), membranes (e.g., nylon membranes). The support can be made from a wide variety of materials, The support may be biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, discs, beads, cells, membranes, etc., or combinations thereof. The support may have any convenient shape, such as disc, square, circle, etc. The support is preferably flat, but may take on a variety of alternative surface configurations. For example, the support may contain raised or depressed regions on which the attachment takes place. The support preferably forms a rigid support on which to carry out the method described herein. [0062] The support has an array of positions each suitable for attachment of a GFP of the present invention. A linker or surface, suitable for coupling a GFP of the present invention to the solid support at each of the array positions is provided on the support. In particular, immobilization of the GFP in this method of the present invention to a solid support involves conventional protein chemistry. As in most protein-interaction assays carried out on a solid support, blocking of nonspecific protein binding sites is recommended (Engvall et al., "Enzyme-linked Immunosorbent Assay (ELISA). Quantitative Assay of Immunoglobulin G," Immunochemistry 8:871-875 (1971); (Suzuki et al., "Chemiluminescent Microtiter Method for Detecting PCR Amplified HIV- 1

DNA," J. Virol. Methods 38:113-122 (1992), which are hereby incorporated by reference in their entirety). Blocking may be done both prior to applying the protein molecule to the surface (pre-blocking) and repeated following immobilization. An appropriate grade of bovine serum albumin (BSA) for blocking is an exemplary blocking agent. Grade and concentration of BSA may vary, depending on the surface composition, (e.g., whether it is polystyrene, glass, or nitrocellulose membrane). Suitable alternatives include poly-L-lysine, nonfat milk, gelatin, or serum. Buffers suitable for dilution of the blocking agent include phosphate buffered saline (PBS) and tris- buffered saline (TBS) (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), which is hereby incorporated by reference in its entirety). These buffers are also suitable for preparation of the GFP and target molecules of this aspect of the present invention. Following blocking of the support surface, the GFP molecule is applied to the support surface, followed by a wash step with a suitable buffer and a second blocking step to remove any potential excess blocking capacity created by over-coating of the surface. Pre-treated supports suitable for use in this aspect of the present invention are also commercially available. These include, without limitation, poly-L-lysine and BSA coated, and epoxy and hydrogen-epoxy activated slides. Manufacturer's instructions for immobilization should be followed for commercial supports.

[0063] Yet another aspect of the present invention relates to a microarray comprising a plurality of green fluorescent protein molecules immobilized at particular sites on a support, wherein each green fluorescent protein molecule comprises a different affinity binding peptide tag inserted within a β-barrel structure of the green fluorescent protein.

[0064] Techniques for determining fluorescence for all aspects of the present invention include, without limitation, flow cytometry, fluorescent microscopy, and FRET. These and other techniques are well known in the art, and will not be described in detail herein.

[0065] The multifunctional GFP molecule of the present invention can also be used to develop metal binding sensors for detection and adsorption of heavy metal ions. In particular, the multifunctional GFP may be immobilized to a solid support and a sample containing a metal (e.g., metal contaminated waste) may be contacted with the support, in the method as described above. Alternatively, the multifunctional GFP may be targeted to a cell surface, such as E. coli. Since recombinant E. coli can be grown in large amounts and within a short period of time, a viable metal bioremediation technology is possible.

EXAMPLES

Example 1 — Modification of mgfp5-ER Sequence

[0066] An mgfp5-ER insert was obtained by digestion of pBιN-mgfp5-ER with Bam I-Sacϊ and ligated into the respective sites of pBluescript SK

(Stratagene, La Jolla, CA). The sequence was modified through a number of polymerase chain reaction (PCR) amplifications using Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad, CA) and oligonucleotides for site-directed mutagenesis and insertion of a poly-histidine sequence to produce the GFP 172 variant. The 6 histidines in GFP 172 are shown in bold in Figure 2. Ncol and Sacl sites at the Ν- and C-terminus are underlined. The S65T mutation in the chromophore (TYG) is shown. Since an Nc l site was introduced at the Ν- terminus, an internal Nc l site was removed by a single base change without changing the codon (boxed). Based on deletion analysis, the minimal domain required for GFP fluorescence is amino-acids 7-229 (Li et al, "Deletions of the Aequorea Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence," J of Biol. Chem. 272(45):28545-28549 (1997), which is hereby incorporated by reference in its entirety). Therefore, any changes at the N- or C- terminus will not effect fluorescence. All mutations shown above have been named with reference to the original mGFP5 (Hasellof et al., "Removal of A Cryptic Intron and Subcellular Localization of Green Fluorescent Protein Are Required to Mark Transgenic Arabidopsis Plants Brightly," Proc. Natl. Acad. Sci. USA 94:2122-2127 (1997), which is hereby incorporated by reference in its entirety). [0067] For insertion of the poly-histidine sequence between amino acids 172 and 173 of the mgfp5-ER, a two-step PCR strategy was used. The N-terminal fragment (amino acids 1 -172) was amplified by primer A (T7 primer) and Primer H (5'- TTC GAT GTT GTG GCG GGT CTT G -3')(SEQ ID NO: 2). Similarly, a C-terminal fragment (amino acids 168-242 including the -HDEL terminal sequence) was amplified by primer E (5'-CGC CAC AAC ATC GAA CAC CAT CAC CAT CAC CAT GAC GGC GGC GTG CAA CTC GC -3 ') (SEQ ID NO: 3) to incorporate a stretch of six histidine residues (bold) and a region to overlap with the N-terminal fragment (underlined region which encodes amino acids 168-172) and primer (T3 primer). The amplified fragments were purified by agarose gel electrophoresis and used together to amplify the entire mgfp5-ER using primers A (T7 primer) and J (T3 primer). An internal Ncol restriction site was removed by a silent mutation (changing a T to A) using the two-step PCR strategy. The Ν- terminal fragment was amplified using primers A (T7 primer) and F (5'- GTG TTG GCC AAG GAA CAG GTA -3') (SEQ ID NO: 4) whereas the C-terminal fragment was amplified using primers C (5'- TAC CTG TTC CTT GGC CAA CAC -3')(SEQ ID NO: 5) and J (T3 primer). The purified fragments were used together to amplify the entire mgfp5-ER using primers A (T7 primer) and J (T3 primer). Using the same two-step PCR strategy, serine in position 65 was changed to threonine (S65T). The N-terminal fragment was amplified using primers A (T7 primer) and G (5'- GAA CAC CAT AAG TGA AAG TAG TG - 3'XSEQ ID NO: 6) whereas the C-terminal fragment was amplified using primers D (5'- CAC TAC TTT CAC TTA TGG TGT TC -3*)(SEQ ID NO: 7) and J (T3 primer). The purified fragments were used together to amplify the entire mgfp5- ER using primers A (T7 primer) and J (T3 primer). One final amplification of the mgfp5-ER(S65T) insert was done using primer B (5'- GGC AGG AGG AAC CAT GGC TAG CAA AGG AGA AGA ACT TTT CAC TGG AG -3') (SEQ ID NO: 8) to incorporate an N-terminal methionine (bold), an Ncol site (underlined) and removal of the Arabidopsis thaliana chitinase signal peptide and primer J (T3 primer). The amplified fragment was digested with Ncol-Sacl and ligated into the respective sites of pGEM-5Zf (Promega, Madison, WI). The product from translation of this modified gene will be referred to as GFP 172 (see Figure 2). All modifications to the sequence were verified by DΝA sequencing. A similar strategy was used to generate GFP 157 by inserting a stretch of six histidine residues between Glnl57 and Lysl58.

[0068] The control, mGFP5-ER(S65T) with 6xHis in the C-terminus

(GFPHis) was made using the primer B as sense and 5' -CGG GCA GAG CTC TTA ATG GTG ATG GTG ATG GTG AAG CTC ATC ATG TTT GTA TAG TTC- 3' (SEQ ID NO: 9) as antisense primers to retain Sad (underlined) and insert a 6xHis tag (bold).

Example 2 ~ MBP-GFP172 Fusion Protein

[0069] The gene coding for GFP 172 was modified by PCR in order to fuse it to the C-terminus of Maltose Binding Protein. The Ncol site in the N-terminal of GFP 172 was modified to an EcoRI site (underlined) using 5 ' - CGG CCG AAT TCA GTA AAG GAG AAG AAC TTT TCA CTG GAG- 3' (SEQ ID NO: 10) as sense and Sad site was modified to a Xbal (underlined) using 5' -CGG GCA GAT CTA GAT TAA AGC TCA TCA TGT TTG TAT AG -3' (SEQ ID NO: 11) as an antisense primer. The amplified PCR product was purified using GENECLEAN (BIOIOI Systems, Carlsbad, CA) gene clean kit. The cleaned product was digested with EcoRI and Xbal, purified by agarose gel electrophoresis, and ligated into the respective sites of pMAL-c2x vector (New England Biolabs, Beverly, MA). The product from translation of this modified gene is referred to as MBP-GFP172.

Example 3 — Expression in E. coli [0070] The modified mgfp5-ER(S65T) inserts were obtained by digestion with Ncoϊ-Sacl and ligated into the respective sites of pET-21d vector (Novagen, Madison, WI). The resulting plasmids were transformed into E. coli BL21trxE(DE3) competent cells (Novagen, Madison, WI) following the manufacturer's instructions. Transformants containing the GFP constructs produced yellow-green colonies. Cultures were grown at the appropriate temperature (37°C or 28°C) in Luria-Bertani (LB) media containing 100 μg/ml ampicillin to an OD₆₀₀ of approximately 0.6 and induced with 1 mM isopropyl- beta-D-thiogalactopyranoside (IPTG) for 4 hours. The cells were harvested by centrifugation for 15 minutes at 3000xg at 4°C and resuspended in 50 mM

NaH₂PO (pH 7.0), 300 mM NaCl. Lysozyme was added to a final concentration of 0.75 mg/ml and the resuspended cells were incubated at room temperature for 30 minutes. The sample was sonicated 6 x 20 seconds on ice at a minimum power setting using an ultrasonic cell disrupter equipped with a microprobe (Branson Model 250, Danbury, CT). The lysate was centrifuged for 20 minutes at 12,000xg at 4°C and soluble GFP in the supernatant was purified by immobilized metal affinity chromatography (IMAC) using TALON cobalt resin columns (Clontech, Palo Alto, CA) according to the manufacturer's instructions.

Example 4 — Protein Assay [0071] Protein was determined using the Micro BCA Protein Assay

Reagent (Pierce Chemical Co., Rockford, IL) with bovine serum albumin or purified recombinant GFP protein (Clontech, Palo Alto, CA) as the standard.

Example 5 — Analysis of Proteins

[0072] Protein samples were denatured in SDS sample buffer containing 2-mercaptoethanol for 3 minutes at 100°C and resolved on a 12% SDS- polyacrylamide gel and a 10% gel in the case of the fusion protein as described (Laemmli 1970). Gels were stained with Coomassie brilliant blue. [0073] For comparison of soluble and insoluble protein following lysis of

E. coli, a sample from the supernatant (cytosolic) was mixed with SDS loading sample buffer whereas a sample of the pellet (insoluble fraction) was dissolved in SDS loading sample buffer with volumes adjusted such that each lane in the gel represented 100 μl of original E. coli cell culture.

[0074] To observe fluorescence of the fusion protein, samples were prepared in SDS sample buffer without mercaptoethanol and directly loaded on the gel without boiling.

Example 6 — Fluorescence Measurements

[0075] GFP fluorescence was measured using a fluorescence spectrophotometer (Hitachi Model F-2500, Tokyo, Japan). Wavelength scans were performed on all GFP variants to determine excitation and emission peaks. Unless otherwise stated, the fluorescent intensity per unit protein was determined with the GFP samples diluted in GFP buffer (50 mM NaH₂PO₄ (pH 8.0), 10 mM Tris-HCl (pH 8.0), 200 mM NaCl) with excitation set at 490 mn and emission set at 512 nm.

Example 7 « Percentage Recovery of GFP by IMAC [0076] The recovery of GFP by having polyhistidine in the loop was compared to having the His tag on the C-terminus. The E. coli derived protein was quantified by BCA Protein Assay (Pierce, Rockford, IL). A total of 20 μg of protein was made up to 1 ml by the addition of extraction buffer (50mM NaH₂PO₄, 300mM NaCl at pH 7.0) and fluorescence measured with excitation set at 490 nm and emission at 512 nm. The protein solution was allowed to pass through equilibrated TALON cobalt resin columns (Clontech, Palo Alto, CA) by gravity. The flow through was collected and its fluorescence was measured in the fluorescence spectrophotometer. After washing the column according to the manufacturer's instructions, the protein was eluted with 3 ml of elution buffer (50mM NaH₂PO₄, 300mM NaCl, 150 mM Imidazole at pH 7.0) and collected in lml fractions. Intensity measurements were made for the eluted samples as described earlier. The intensity measurements were used to estimate the amount of GFP present in the eluted fractions by standard curves of fluorescence vs amount of protein (μg/ml) made for the purified protein. Example 8 - pH Titration

[0077] Fluorescence intensities of GFP (20μg/ml) in a buffer (Kneen et al., "Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator," Biophysical Journal 74:1591-1599 (1998), which is hereby incorporated by reference in its entirety) containing 120mM KC1, 5mM NaCl, 0.5mM CaCl₂, 0.5mM MgSO₄, lOmM MES, lOmM MOPS and lOmM Citrate at pH values ranging from 8.0 to 5.0 differing by 0.5 pH units, was measured in a quartz cuvette. The pH was adjusted using IN HC1. Emission intensities were plotted as a function of pH and curve fitted (Kneen et al., "Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator," Biophysical Journal 74:1591-1599 (1998), which is hereby incorporated by reference in its entirety).

Example 9 — Tobacco Extract and Spiking Studies

[0078] Tobacco leaves from wild-type potted plants were ground in liquid nitrogen with a mortar and pestle until a fine powder was obtained. The powder was resuspended in extraction buffer (50mM NaH₂PO₄, 300mM NaCl pH 7.0) and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected and filtered using a Whatman 70mm filter paper to remove floating solid particles that remained after centrifugation. The total protein in the extract was quantified by the Biorad protein assay using bovine serum albumin (BSA) as standard. ImM phenylmethyl sulfonyl fluoride (PMSF) was added to the extract to inhibit protease activity. The MBP-GFP172 fusion protein was spiked into this extract at a concentration representing 0.5% total soluble protein. The fusion protein was purified using TALON (Clontech, Palo Alto, CA) cobalt resin columns and percentage recovery was calculated for the fusion protein as described earlier from a standard series of MBP-GFP172 purified from E. coli lysate.

Example 10 ~ Factor Xa Cleavage

[0079] Factor Xa, a restriction endoprotease, recognizes the amino acid sequence Ile-Glu/Asp-Gly-Arg, and cleaves the peptide bond C-terminal of the arginine residue. It is commonly used to cleave His-tags from peptides in applications where the removal of a protein's His tag is desired. Factor Xa at concentrations of 200ng and 500ng was used to cut 20 μg of a lμg/μl solution of MBP-GFP172 in elution buffer (50mM NaH₂PO₄, 300mM NaCl, 150mM Imidazole pH 7.0) at room temperature. 5 μl samples from this reaction mixture were collected at different time intervals to monitor cleavage by SDS-PAGE analysis.

Example 11 - Effect of S65T Mutation

[0080] A 6xHis tag was inserted at amino acid positions 172-173 and 157-

158 of mGFP5-ER(S65T) and the properties of these new GFPs were evaluated. For the GFPs with the internal His tag, an increase in fluorescence intensities of about 1.4 times was seen, as a result of the S65T mutation, as shown in Figure 3. These GFPs were produced at 37 °C. Cos-7 cells expressing GFP5(S65T) fluoresced 1.65 times higher than cells expressing GFP5 (Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996), which is hereby incorporated by reference in its entirety). However, the chromophore in S65T type GFPs is known to be effected by temperature (Patterson et al., "Use of the Green Fluorescent Protein and its Mutants in Quantitative Fluorescence Microscopy," Biophysical Journal 73:2782-2790 (1997), which is hereby incorporated by reference in its entirety).

Example 12 ~ Effect of Temperature on GFP Expression

[0081] Effect of temperature on GFP expression showed a significant difference in the amount of soluble GFP produced at 28°C and 37°C for the two loop GFPs. Soluble GFP 172 was obtained at both 37°C, as shown in Figure 4A, Lane 3, and 28°C, shown in Figure 4B, Lane 3. In the case of GFP157, most of the protein is insoluble at the two temperatures with almost no soluble protein at 37°C, shown in Figure 4A, Lane 5. However, the GFP with the C-terminal His tag, expressed significant amounts of soluble protein at both temperatures. It has been shown in the past that proteins expressed at 28°C were much brighter than that expressed at 37°C in the case of wt-GFP and S65T and this was attributed to a permanent chromophore deformation at 37°C (Patterson et al., "Use of the Green Fluorescent Protein and its Mutants in Quantitative Fluorescence Microscopy," Biophysical Journal 73 :2782-2790 (1997), which is hereby incorporated by reference in its entirety). When expressed at 28°C, it was shown that the chromophores formed efficiently in many variants of GFP (Patterson et al., "Use of the Green Fluorescent Protein and its Mutants in Quantitative Fluorescence Microscopy," Biophysical Journal 73:2782-2790 (1997), which is hereby incorporated by reference in its entirety). GFP5 was designed to be a thermostable folding mutant with enhanced spectral characteristics (Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996), which is hereby incorporated by reference in its entirety). It was shown that the post-translational oxidation to form the mature GFP chromophore is not the step responsible for the temperature sensitivity instead it is primarily due to mis-folding of GFP at elevated temperatures. Moreover, Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996), which is hereby incorporated by reference in its entirety, found that His tag at the C-terminus has minimal effects on this folding property at higher temperatures. In the case of GFP 172 and GFP 157, the lower temperature is favored for expression of soluble protein and this could be because of mis-folding at the elevated temperature. In the case of GFP 157, the effect of the internal His tag in misfolding at the higher temperature is more prominent than for GFP 172. GFP expressed in Saccharomyces cerevisiae were less fluorescent at high culture temperatures (Lim et al., "Thermosensitivity of Green Fluorescent Protein

Utilized to Reveal Novel Nuclear-Like Compartments in a Mutant Nucleophorin NSP1," J. Biochemistry (Tokyo) 118:13-17 (1995), which is hereby incorporated by reference in its entirety). By adding the Serl47 to Pro (S147P), this folding problem at higher temperature could probably be overcome. It was shown that when S147P was combined with S65T, the resulting double mutant emitted higher fluorescence than GFP with a single S65T mutation and could be used over a wide range of culturing temperatures (Kimata et al., "A Novel Mutation that Enhances the Fluorescence of Green Fluorescent Protein at High Temperatures," Biochem. Biophys. Res. Commun. 232(l):69-73 (1997), which is hereby incorporated by reference in its entirety). Example 13 — Fluorescence Intensity

[0082] A comparison of fluorescence intensities produced at different temperatures is shown in Figure 5. The fluorescence intensity of GFP 172 produced at 28°C was 65% of GFPHis, but was brighter than the GFP 172 produced at 37°C. The intensity of GFP 157 produced at 28°C was approximately the same as that of GFP 157 at the same temperature (Figure 5). The intensities have been normalized with respect to GFPHis. It was found that the GFPs produced at 28°C were brighter, which is consistent with the explanation that chromophore formation is more efficient at 28°C, and with the fact that the fraction of completely folded GFP may be higher at the lower temperature. Moreover, the amount of light absorbed by GFP 172 produced at 37°C as seen from the excitation peak in Figure 6 was 59% of GFPHis. Similarly, the excitation peak for GFP 172 at 28°C is 63% of that absorbed by GFPHis, shown in Figure 7. This reduction in the excitation peak could be because of a change in the microenvironment around the chromophore resulting in less molar absorbance of incident light and, hence, decrease in emission by similar amounts. Affinity fluorescent proteins were made by Matsudaira et al., "Affinity Fluorescent Proteins and Uses Thereof. International Patent," Publication number WO 01/09177 A2 (2001), which is hereby incorporated by reference in its entirety, by inserting an epitope for haemagglutinin at positions 172-173 and 157-158 of GFP. It was seen that the 395 nm peaks were 40% and 33.3% respectively compared to wt-GFP. When excited at 395 nm, the emission peaks at 550 mn were 40% and 32.8% for 172-173 and 157-158 residues compared to wt-GFP. An interesting note is that the 395 nm peak, characteristic of a GFP5 variant, is not seen in the excitation scan. This implies that the S65T mutation dominates and, therefore, only a single 490 nm peak is seen, consistent with what was obtained earlier for S65T (Heim et al., "Improved Green Fluorescence," Nature 373:663-664 (1995), which is hereby incorporated by reference in its entirety).

Example 14 — Purity and Recovery [0083] Since the poly-histidine tag in the GFP loop is accessible to the immobilized metal, highly pure samples of GFP 172 and GFP 157 were obtained by IMAC as observed by SDS-PAGE, as shown in Figure 8. For the same reason, even the recovery obtained for GFP 172 (87%) was comparable to that of having the tag in the C-terminus (81%), shown in Figure 9. No fluorescence was seen in the flow-through and the washes, indicating binding of the protein to the column and the minor loss of protein as seen in the recovery could be because of irreversible binding to the column.

Example 15 - Effect of pH

[0084] pH plays a significant role on GFP fluorescence, as can be seen from the titration curve shown in Figure 10. The titration data were fitted to the following equation:

F= 1 / [1+ 10^ΛnH(pKa-pH)] (1)

with parameters pKa (pH at 50% maximum) and Hill coefficient nH (proportional to the slope of fluorescence versus pH at pKa) (Kneen et al., "Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator," Biophysical Journal 74:1591-1599 (1998), which is hereby incorporated by reference in its entirety). Fitted pKa's were found to be 5.6, 5.45 and 5.39 for GFP 172, GFP 157 and mGFP5ER-6xHis. Past reports have shown that the pKa for S65T was ~ 6.0 (Elsliger et al., "Structural and Spectral Response of Green Fluorescent Protein Variants to Changes in pH," Biochemistry 38:5296-5301 (1999); and Kneen et al., "Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator," Biophysical Journal 74:1591-1599 (1998), which are hereby incorporated by reference in their entirety). The difference in the pKa obtained can be attributed to the mGFP5-ER used as a template to insert the His tag which has the I167T, V163A and S175G mutations (Heim et al., "Wavelength Mutations and Posttranslational Autooxidation of Green Fluorescent Protein," Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Siemering et al., "Mutations that Suppress the Thermosensitivity of Green Fluorescent Protein," Current Biology 6:1653-1663 (1996), which are hereby incorporated by reference in their entirety). Single point mutations have known to change pKa values drastically. For example, mutagenesis was used to increase the pKa of S65T from 6.0 to 7.8 (S65T/H148D) presumably due to the proximity of the negatively charged aspartate (Elsliger et al., "Structural and Spectral Response of Green Fluorescent Protein Variants to Changes in pH," Biochemistry 38:5296-5301 (1999), which is hereby incorporated by reference in its entirety). EGFP (F64L/S65T) is 50% quenched at approximately pH 5.5 (Patterson et al., 1996). [0085] It can be clearly seen from the pKa values obtained that the position of the His tag in the GFP has minimal effects on the GFP pKa. Kneen and coworkers (Kneen et al., "Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator," Biophysical Journal 74:1591-1599 (1998), which is hereby incorporated by reference in its entirety) have shown by spectroscopic and kinetic studies that the fluorescence of GFP decreased because of a change in the molar absorbance as a result of simple protonation-deprotonation of residues for pH > 5.0, whereas below pH 5.0 the GFP unfolds. Similar results were obtained where the shape of the spectra didn't change with pH, but there was a decrease in molar absorbance. Crystallographic studies have shown that phenolic hydroxyl of the chromophore is the titrating group responsible for the pH sensitive mechanism of S65T (Elsliger et al., "Structural and Spectral Response of Green Fluorescent Protein Variants to Changes in pH," Biochemistry 38:5296-5301 (1999); Kneen et al., "Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator," Biophysical Journal 74:1591-1599 (1998), which are hereby incorporated by reference in their entirety) and protonation state of the imidazolinone ring nitrogen unchanged (Elsliger et al., "Structural and Spectral Response of Green Fluorescent Protein Variants to Changes in pH," Biochemistry 38:5296-5301 (1999), which is hereby incorporated by reference in its entirety). Use of GFP as an intracellular pH indicator has been demonstrated (Llopsis et al., "Measurement of Cytosolic, Mitochondrial, and Golgi pH in Single Living Cells with Green Fluorescent Proteins," PNAS 95 :6803-6808 (1998); Kneen et al., "Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator," Biophysical Journal 74:1591-1599 (1998), which are hereby incorporated by reference in their entirety). These pH titration experiments will be useful to make appropriate corrections and calibrations in fluorescence values depending on the organelle/cytosol where the dual-functional tag fused to a target protein is produced which, in turn, will depend on the N- and/or C-terminal targeting and retention signals. Moreover, depending on the pH environment of the expressed fusion protein, appropriate GFP mutants with desirable pKa values can be used as a template for the insertion of a His tag at the positions selected herein, with the assumption that the tag will not have drastic effects on the titrating group of the chromophore.

Example 16 - MBP-GFP172 Fusion Protein [0086] On comparing the various parameters analyzed, the only significant difference between GFP 172 and GFP 157 is that the former could be produced in soluble form at both 37°C and 28°C, shown in Figures 3A-B. Based on these preliminary experiments, GFP 172 was fused to maltose binding protein (MBP) and the fusion protein was expressed in E. coli BL21(DE3) trxB cells (Novagen, Madison, WI). The ability to remain soluble at both 28°C and 37°C allows use of the tag in a broad host range including mammalian cells, plant cells, yeast, and bacteria, including E. coli. Numerous applications of fusion proteins are reported in the literature. In essence, fusion proteins have been made either for providing affinity purification tags, for enhancing the solubility of the fusion partner, or for suppressing degradation. The solubilizing ability of commonly used fusion partners like maltose-binding protein (MBP), glutathione S-transferase (GST) and thioredoxin (TRX) were compared by fusing six diverse proteins that normally accumulate in insoluble form. It was found that MBP was the most effective fusion partner (Kapust et al., "Escherichia coli Maltose-Binding Protein is Uncommonly Effective at Promoting the Solubility of Polypeptides to Which it is Fused Protein," Science 1668-1674 (1999), which is hereby incorporated by reference in its entirety). MBP can also be used to purify the fusion partner by immobilized amylose resin affinity chromatography (Riggs, "Current Protocols in Molecular Biology" (Ausubel FM et al, 1990) pp. 16.4.1/16.6.1. Greene Associates/Wiley Interscience, New York, which is hereby incorporated by reference in its entirety). An MBP-GFPuv fusion protein was made to study protein localization with and without the MBP signal sequence using GFPuv as a reporter (Feilmeier et al., "Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli," Journal of Bacteriology pp. 4068-4076 (2000), which is hereby incorporated by reference in its entirety). It was found that the fusion protein localized to the cytoplasm fluoresced, but that localized to the periplasmic space did not. Solubility and folding of the fusion partner is also influenced by the position of MBP in the fusion, as was demonstrated with procathepsin D and pepsinogen, both being proteins that normally form inclusion bodies. However, MBP in the N-terminus resulted in the fusion protein being soluble in the bacterial cytosol and was able to bind to amylose resin, but formed inclusion bodies if fused to the C-terminus (Sachdev et al., "Order of Fusions Between Bacterial and Mammalian Proteins Can Determine Solubility in Escherichia coli," Biochem. Biophvs. Res. Commun. 244:933 (1998), which is hereby incorporated by reference in its entirety). [0087] The GFP 172 tag was fused to the C-terminus of maltose binding protein and expressed in E. coli BL21(DΕ3) trxB cells (Novagen, Madison, WI) in soluble form to test the ability of the GFP tag to serve its dual functions as a fusion protein. Figure 11 (Lane 2) shows that the GFP 172 with the internal 6xHis tag can be used to purify the fusion protein to homogenous purity in a single step from a crude E. coli lysate. This implies that the 42.5 KDa MBP attached to GFP 172 does not interfere with the accessibility of the internal 6xHis tag to the immobilized metal. As mentioned earlier, this accessibility is due to the presence of the 6xHis tag in the solvent accessible loop of GFP. The purified fusion protein was soluble and fluoresced, as determined by SDS-PAGE gel, shown in Figure 12, for which the samples were prepared in loading buffer without any reducing agent and loaded without boiling so that the fusion protein was maintained in its native state. Moreover, it has been established that GFP fluoresces even in the presence of 1% SDS (Bokman et al., "Renaturation of Aequorea Green Fluorescent Protein," Biochem. Biophys. Res. Commun. 101: 1372-1380 (1981), which is hereby incorporated by reference in its entirety) and is resistant to acrylamide (Patterson et al., "Use of the Green Fluorescent Protein and its

Mutants in Quantitative Fluorescence Microscopy," Biophysical Journal 73:2782- 2790 (1997), which is hereby incorporated by reference in its entirety). Since the GFP fluoresces even as a fusion protein, it can be concluded that the MBP is also active because full length protein is produced. This can be seen from the size of approximately 71 kDa on the gel shown in Figure 11, and from the fact MBP is fused to the N-terminus of GFP 172. As mentioned earlier, position of MBP in the fusion influences the solubility of the fusion partner. Moreover, MBP-GFP (Kapust et al., "Escherichia coli Maltose-Binding Protein is Uncommonly Effective at Promoting the Solubility of Polypeptides to Which it is Fused Protein," Science 1668-1674 (1999), which is hereby incorporated by reference in its entirety) and MBP-GFPuv (Feilmeier et al., "Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli," Journal of Bacteriology pp. 4068-4076 (2000), which is hereby incorporated by reference in its entirety) fusion proteins were reported to be soluble.

Example 17 — Spiking Studies

[0088] In order to test if the GFP tag was capable to purify the fusion protein from plant extracts, MBP-GFP172 was spiked into tobacco leaf extract and analyzed for the ability of the tag to recover the fusion protein. A concentration of 0.5% total soluble protein was used for spiking, which is representative of the level of expression achieved for GFP in plants from past work. Expression of mGFP5-ER driven by Cauliflower Mosaic Virus (CaMV) 35S promoter ranged up to 7.05 μg GFP per mg of extractable protein (0.7%)

(Remans et al., "A Protocol for the Fluorimetric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants," Plant Molecular Biology Reporter 17:385-395 (1999), which is hereby incorporated by reference in its entirety). GFP expression levels in tobacco ranging from 0.0-0.5% of total extractable protein has been reported and a minimal amount of approximately 0.1% GFP was required for unambiguous macroscopic detection (Leffel et al., "Applications of Green Fluorescent Protein in Plants," Biotechniques 23(5):912-918 (1997), which is hereby incorporated by reference in its entirety). The transgenic tobacco plants grown under artificial light driven by CaMV 35S promoter were analyzed for GFP expression patterns. GFP synthesis levels varied between 0.12% to 0.15% in leaves (Harper et al., "Patterns of Green Fluorescent Protein Expression in Transgenic Plants," Plant Molecular Biology Reporter 18:141a-141i (2000), which is hereby incorporated by reference in its entirety). [0089] In spiking and recovery experiments the average recovery was about 75% and purity as judged by SDS-PAGE was greater than 75%, as determined by scanning the Coomassie stained gel, shown in Figure 13, Lane 3, and densitometry analysis using SigmaScan Pro 5.0 image-analysis software (SPSS Inc., Chicago, IL). The recovery was calculated as described above, from a standard series of the fusion protein purified from E. coli lysate, shown in Figure 14. This methodology has been used in the past where a GFP-standard series was prepared for purified GFP in untransformed plant extract to determine GFP concentration in transgenic plant extracts (Remans et al., "A Protocol for the Fluorimetric Quantification of mGFP5-ΕR and sGFP(S65T) in Transgenic Plants," Plant Molecular Biology Reporter 17:385-395 (1999), which is hereby incorporated by reference in its entirety). Lactate dehydrogenase (LDH) was purified from tobacco extract using a 6xHis tail at the N-terminus by IMAC with a Zn²⁺ chelated gel (Mejare et al., "Evaluation of Genetically Attached Histidine Affinity Tails for Purification of Lactate Cehydrogenase from Transgenic Tobacco," Plant Science 134:103-114 (1998), which is hereby incorporated by reference in its entirety). Enzyme recovery of 55% was obtained for LDHHis₆ compared to 7% for native LDH having no His tail. It was also reported that native LDH did not bind to Co²⁺ similar to results obtained in another work where native β-glucuronidase (GUS) did not bind to Co²⁺ chelated to iminodiacetate

(IDA) (Zhang et al., "Suitability of Immobilized Metal Affinity Chromatography for Protein Purification from Canola," Biotechnology and Bioengineering 68:52- 58 (2000), which is hereby incorporated by reference in its entirety). However, β- glucuronidase-his₆ (GUSH6) was purified from canola protein extract with almost homogenous purity in a single chromatographic step using Co²⁺ with iminodiacetate (IDA) as the chelating ligand. It was found that Co²⁺-IDAhad the least amount of non-specific binding of canola proteins than metals like Cu²⁺, Ni²⁺ and Zn²⁺ and therefore higher purity of the target protein (Zhang et al., "Suitability of Immobilized Metal Affinity Chromatography for Protein Purification from Canola," Biotechnology and Bioengineering 68:52-58 (2000), which is hereby incorporated by reference in its entirety). This highlights the importance of the 6xHis tag in the performance of IMAC compared to the native protein. Just binding to the immobilized metal is not sufficient. There needs to be a preferential binding of the target protein with respect to the numerous other proteins found in the host system. For the same reasons, using Cu(II) to purify GFPuv as was reported by Li et al., "Characterization of Metal Affinity of Green Fluorescent Protein and its Purification through Salt Promoted, Immobilized Metal Affinity Chromatography," J. of Chromatography A 909:183-190 (2001), which is hereby incorporated by reference in its entirety, without a His tail, may not be feasible in most cases for reasons of non-specific binding of host proteins because Cu(II) has the ability to recognize surface histidines. Moreover, the Cu(II) system requires high amounts of NaCl to work effectively (Li et al., "Characterization of Metal Affinity of Green Fluorescent Protein and its Purification through Salt Promoted, Immobilized Metal Affinity Chromatography," J. of Chromatography A 909:183-190 (2001), which is hereby incorporated by reference in its entirety) and that, in turn, would result in hydrophobic interactions causing more non-specific binding. The Cu(II) system would also be ineffective if there was a His tail on the target protein because the binding of the target protein would be too strong, requiring harsh conditions for elution which might disrupt the target protein. This can result in lower recoveries, as was seen in GUSH6 recovery on Cu²⁺-IDA (Zhang et al (1999), which is hereby incorporated by reference in its entirety).

[0090] From the gel in Figure 13, it can see that the purity of the fusion protein (~71 kDa) is quite high. However, a lower band having the size of approximately 32 kDa is seen in the lane in spite of adding the protease inhibitor PMSF, as described above in Example 9. Because no other significant band, apart from that of the fusion protein and this lower band, is seen, it is believed that the lower band is either a product of protease activity on the fusion protein or is a protein from the tobacco extract. The PMSF may not have been effective enough to inactivate protease activity to completion. A western blot using anti-GFP antibody on this fraction does show this lower band and, therefore, rules out the second possibility of the band being a protein from the extract. Since the size of this lower band is approximately the size of GFP, it was necessary to determine if this band is responsible for any fluorescence. Since the recovery data is obtained from a standard series of the fusion protein, fluorescence if any contributed by the lower band will distort the values obtained for recovery. Samples were run on an SDS-PAGE gel without boiling the samples prior to loading on the gel, so that the protein retained its fluorescence. Lane 3 in Figure 15 represents protein recovered from an extract with no PMSF added. Figure 15, Lanes 4 and 5 show protein recovered from extract with ImM PMSF. No fluorescent band around the size of the GFP 172 standard is seen in Lanes 3, 4, or 5, implying that there is no contribution to the fluorescence of the eluted fraction by the lower band. Based on densitometry analysis using SigmaScan Pro 5.0 image-analysis software (SPSS Inc., Chicago, IL) and a comparison of intensity of fusion protein standards on the Coomassie stained gel, shown in Figure 13, the lower bands in Lanes 4 and 5 are estimated to have at least 520ng and 780ng of protein, respectively. However, none, or at most, negligible amounts of this protein fluoresces by comparing to the bands in Lanes 7, 8 and 9, which have different concentrations of purified

GFP 172, the lowest concentration being lOOng in Lane 7. Since no band in Lane 3, 4, or 5 is seen around the size of GFP 172, it can be concluded that the majority of the protein in the lower band is non-fluorescent. The amount of fluorescent protein, if any, would be far less than lOOng, and because Lanes 3 and 4 have 2 μg total protein and Lane 5 has 3 μg total protein, the error would be far less than 5%. Therefore, the fluorescence of the recovered fractions is a good estimate of the amount of fusion protein recovered. The cleavage of the fusion protein would depend on the recombinant protein of interest fused to the tag, the linker, and the host system used for expression. Therefore, the use of protease inhibitors needs to be optimized on a case-by-case basis.

Example 18 - Factor Xa Cleavage

[0091] In a test run to determine Factor Xa cleavage, the enzyme was added in a ratio of 1 : 100 for Factor Xa to fusion protein. As can be seen from gel in Figure 16 A, even after 40 hours, the cleavage of the fusion protein into two distinct bands is not complete. This is probably because the cleavage site is not completely exposed to cleave the fusion protein to completion at the recommended test concentration of Factor Xa. On raising the Factor Xa concentration by 2.5 times, complete cleavage was achieved, shown in Figure 16B, lane 5. Factor Xa concentration cannot be increased to a great extent because secondary cleavage products would be produced. Therefore, an optimum concentration of Factor Xa needs to be determined for each fusion protein with a Ile-Glu/Asp-Gly-Arg Factor Xa recognition site in-between the two proteins. Factor Xa cleaves after Arg and sometimes at other basic residues, depending on the conformation of the protein substrate (Nagai et al., "Generation of Beta- Globin by Sequence-Specific Proteolysis of a Hybrid Protein Produced in Escherichia coli," Nature 309(5971):810-812 (1984); Eaton et al, "Proteolytic Processing of Human Factor VIII. Correlation of Specific Cleavages by

Thrombin, Factor Xa, and Activated Protein C with Activation and Inactivation of Factor VIII Coagulant Activity," Biochemistry 25(2):505-512 (1986), which are hereby incorporated by reference in their entirety). Factor Xa can be removed from the reaction mixture by passing the mixture through benzamidine sepharose resin (Amersham Pharmacia Biotech, Buckinghamshire, England).

[0092] The multifunctional GFP tag of the present invention can be effectively utilized in different host systems. This multifunctional GFP tag can be used to monitor expression of any target protein fused to it, and can also monitor purification and recovery by IMAC, by measuring fluorescence of the fractions. Once the fusion protein is purified, the tag can be removed from the protein of interest by an appropriate choice of linkers.

[0093] It has been demonstrated that by simply measuring GFP fluorescence in E. coli cultures, amounts of active organophosphorous hydrolase (OPH) in the culture could be estimated because GFP was fused to the N-terminus of OPH (Wu et al., "A Freen Fluorescent Protein Fusion Strategy for Monitoring the Expression, Cellular Location, and Separation of Biologically Active Organophosphorus Hydrolase," Appl. Microbiol. Biotechnol. 54(l):78-83 (2000), which is hereby incorporated by reference in its entirety). The GFP fluorescence was also used to track OPH purification by a 6xHis tag on the N-terminus of GFP. In a more recent finding, GFP fused to the N-terminus of μ-opioid receptor

(HuMOR) was used to quantify HuMOR expression levels and the fusion did not effect the expression levels and ligand binding properties of the functional receptor (Sarramegna et al., "Green Fluorescent Protein as a Reporter of Human s-opioi Receptor Overexpression and Localization in the Methylotrophic Yeast Pichia pastoris " Journal of Biotechnology 99:23-39 (2002), which is hereby incorporated by reference in its entirety). However, by having an internal 6xHis tag in the GFP, as demonstrated in the present invention, more flexibility is provided in terms of attaching signal peptides and retention signals to the desired recombinant protein, providing the advantage of better accumulation and stability of the recombinant protein. Moreover, depending on the application and the system in which it is being produced, the His tag may be inserted in other variants of GFP in the positions. Other purification tags could also be inserted in the positions disclosed above. Commercial vectors can be developed to incorporate these features and recombinant proteins could be expressed and monitored in different host organisms and also purified by IMAC.

[0094] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. A multifunctional green fluorescent protein comprising: green fluorescent protein and a purification tag inserted within a β-barrel structure of the green fluorescent protein, wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein.

2. The multifunctional green fluorescent protein according to claim 1 , wherein the green fluorescent protein is selected from the group consisting of a wild-type green fluorescent protein and variants thereof.

3. The multifunctional green fluorescent protein according to claim 2, wherein the variant green fluorescent protein is a mGFP5 variant, a GFP5-S65T variant, a cyan fluorescent protein variant, a yellow fluorescent protein variant, a red fluorescent protein variant, or a blue fluorescent protein variant.

4. The multifunctional green fluorescent protein according to claim 3, wherein the variant green fluorescent protein is the mGFP5 variant.

5. The multifunctional green fluorescent protein according to claim 3, wherein the variant green fluorescent protein is the GFP5-S65T variant.

6. The multifunctional green fluorescent protein according to claim 1, wherein the purification tag is selected from the group consisting of a poly-histidine molecule, a charged amino acid molecule, and elastin-like polypeptides.

7. The multifunctional green fluorescent protein according to claim 6, wherein the purification tag is a poly-histidine molecule.

8. The multifunctional green fluorescent protein according to claim 7, wherein the poly-histidine molecule is a hexa-histidine molecule.

9. The multifunctional green fluorescent protein according to claim 1 , wherein the purification tag is inserted between sites Glnl57-Lysl58 of the green fluorescent protein.

10. The multifunctional green fluorescent protein according to claim 1 , wherein the purification tag is inserted between sites Glul72-Aspl73 of the green fluorescent protein.

11. A fusion protein comprising: a green fluorescent protein having a purification tag inserted within a β- barrel structure of the green fluorescent protein, wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, and at least one protein or polypeptide operably linked to the green fluorescent protein.

12. The fusion protein according to claim 11 , wherein the green fluorescent protein is selected from the group consisting of a wild-type green fluorescent protein and variants thereof.

13. The fusion protein according to claim 12, wherein the variant green fluorescent protein is a mGFP5 variant, a GFP5-S65T variant, a cyan fluorescent protein variant, a yellow fluorescent protein variant, a red fluorescent protein variant, or a blue fluorescent protein variant.

14. The fusion protein according to claim 13, wherein the variant green fluorescent protein is the mGFP5 variant.

15. The fusion protein according to claim 13 , wherein the variant green fluorescent protein is the GFP5-S65T variant.

16. The fusion protein according to claim 11 , wherein the purification tag is selected from the group consisting of a poly-histidine molecule, a charged amino acid molecule, and elastin-like polypeptides.

17. The fusion protein according to claim 16, wherein the purification tag is a poly-histidine molecule.

18. The fusion protein according to claim 17, wherein the polyhistidine molecule is a hexa-histidine molecule.

19. The fusion protein according to claim 11 , wherein the purification tag is inserted between sites Glnl57-Lysl58 of the green fluorescent protein.

20. The fusion protein according to claim 11, wherein the purification tag is inserted between sites Glul 72-Asp 173 of the green fluorescent protein.

21. A nucleic acid construct comprising: a first nucleic acid molecule encoding a green fluorescent protein according to claim 1 operably linked to at least one second nucleic acid molecule encoding at least one protein or polypeptide, wherein the first and at least one second nucleic acid molecules are operably linked to 5' and 3' regulatory nucleic acid molecules that allow expression of the first and at least one second nucleic acid molecules, and wherein expression of the first and at least one second nucleic acid molecules produces a green fluorescent protein-at least one protein or polypeptide fusion protein.

22. An expression system comprising a vector into which is inserted the nucleic acid construct according to claim 21.

23. A host cell comprising the nucleic acid construct according to claim 22.

24. The host cell according to claim 23, wherein the host cell is a bacterial cell, a yeast cell, a plant cell, or a mammalian cell.

25. A method for purifying a protein or polypeptide comprising: providing a fusion protein comprising a green fluorescent protein having a purification tag inserted within a β-barrel structure of the green fluorescent protein operably linked to at least one protein or polypeptide; contacting the fusion protein with a substrate having a binding affinity for the purification tag under conditions effective to bind the purification tag to the substrate; and isolating the at least one protein or polypeptide from the bound fusion protein.

26. The method according to claim 25 further comprising: monitoring protein or polypeptide purification by detecting green fluorescent protein fluorescence.

27. The method according to claim 25, wherein the green fluorescent protein is selected from the group consisting of a wild-type green fluorescent protein and variants thereof.

28. The method according to claim 27, wherein the variant green fluorescent protein is a mGFP5 variant, a GFP5-S65T variant, a cyan fluorescent protein variant , a yellow fluorescent protein variant, a red fluorescent protein variant, or a blue fluorescent protein variant.

29. The method according to claim 28, wherein the variant green fluorescent protein is the mGFP5 variant.

30. The method according to claim 28, wherein the variant green fluorescent protein is the GFP5-S65T variant.

31. The method according to claim 25, wherein the purification tag is selected from the group consisting of a metal-binding peptide, a carbohydrate-binding domain, a biotin-binding domain, an antigenic epitope, a nucleic acid-binding peptide, and a peptide that binds to a molecularly-imprinted polymer.

32. The method according to claim 31 , wherein the purification tag is a metal-binding peptide.

33. The method according to claim 32, wherein the metal-binding peptide is a poly-histidine molecule.

34. The method according to claim 33, wherein the poly-histidine molecule is a hexa-histidine molecule.

35. The method according to claim 25, wherein the purification tag is inserted between sites Glnl57-Lysl58 of the green fluorescent protein.

36. The method according to claim 25, wherein the purification tag is inserted between sites Glul72-Aspl73 of the green fluorescent protein.

37. A method for purifying a protein or polypeptide comprising: providing a fusion protein comprising a green fluorescent protein having a purification tag inserted within a β-barrel structure of the green fluorescent protein operably linked to at least one protein or polypeptide, wherein the purification tag is an amino acid sequence that introduces a change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein, and isolating the at least one protein or polypeptide from the fusion protein based on the change in charge, electrophoretic mobility, hydrophobicity, solubility, or partitioning to the green fluorescent protein.

38. The method according to claim 37 further comprising: monitoring protein or polypeptide purification by detecting green fluorescent protein fluorescence.

39. The method according to claim 37, wherein the green fluorescent protein is selected from the group consisting of a wild-type green fluorescent protein and variants thereof.

40. The method according to claim 39, wherein the variant green fluorescent protein is a mGFP5 variant, a GFP5-S65T variant, a cyan fluorescent protein variant , a yellow fluorescent protein variant, a red fluorescent protein variant, or a blue fluorescent protein variant.

41. The method according to claim 40, wherein the variant green fluorescent protein is the mGFP5 variant.

42. The method according to claim 40, wherein the variant green fluorescent protein is the GFP5-S65T variant.

43. The method according to claim 37, wherein the purification tag is selected from the group consisting of a poly-histidine molecule, a charged amino acid molecule, and elastin-like polypeptides.

44. The method according to claim 43, wherein the purification tag is a poly-histidine molecule.

45. The method according to claim 44, wherein the poly-histidine molecule is a hexa-histidine molecule.

46. The method according to claim 37, wherein the purification tag is inserted between sites Glnl57-Lysl58 of the green fluorescent protein.

47. The method according to claim 37, wherein the purification tag is inserted between sites Glul72-Aspl73 of the green fluorescent protein.

48. A method for identifying expression of a protein or polypeptide in biological material comprising: providing a nucleic acid construct comprising: a first nucleic acid molecule encoding a green fluorescent protein according to claim 1 operably linked to at least one second nucleic acid molecule encoding at least one protein or polypeptide, wherein the first and at least one second nucleic acid molecules are operably linked to 5' and 3' regulatory nucleic acid molecules that allow expression of the first and at least one second nucleic acid molecules, and wherein expression of the first and at least one second nucleic acid molecules produces a green fluorescent protein-at least one protein or . polypeptide fusion protein; introducing the nucleic acid construct to a biological material under conditions effective to allow expression of the fusion protein in the biological material; and determining fluorescence of the green fluorescent protein in the biological material to identify the expression of the at least one protein or polypeptide in the biological material.

49. The method according to claim 48, wherein the green fluorescent protein is selected from the group consisting of a wild-type green fluorescent protein and variants thereof.

50. The method according to claim 49, wherein the variant green fluorescent protein is a mGFP5 variant, a GFP5-S65T variant, a cyan fluorescent protein variant, a yellow fluorescent protein variant, a red fluorescent protein variant, or a blue fluorescent protein variant.

51. The method according to claim 50, wherein the variant green fluorescent protein is the mGFP5 variant.

52. The method according to claim 50, wherein the variant green fluorescent protein is the GFP5-S65T variant.

53. The method according to claim 48, wherein the purification tag is selected from the group consisting of a poly-histidine molecule, a charged amino acid molecule, and elastin-like polypeptides.

54. The method according to claim 53, wherein the purification tag is a poly-histidine molecule.

55. The method according to claim 54, wherein the poly-histidine molecule is a hexa-histidine molecule.

56. The method according to claim 48, wherein the purification tag is inserted between sites Glnl57-Lysl58 of the green fluorescent protein.

57. The method according to claim 48, wherein the purification tag is inserted between sites Glul72-Aspl73 of the green fluorescent protein.

58. The method according to claim 48, wherein the biological material is a cell.

59. The method according to claim 58, wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, a plant cell, and a mammalian cell.

60. The method according to claim 59, wherein the cell is a plant cell.

61. The method according to claim 59, wherein the cell is a mammalian cell.

62. The method according to claim 48, wherein the biological material is tissue, bone, or a body fluid.

63. A method for identifying a target molecule comprising: providing a support having a plurality of green fluorescent protein molecules immobilized at particular sites on the support, wherein each green fluorescent protein molecule comprises a different affinity binding peptide tag inserted within a β- barrel structure of the green fluorescent protein; providing a sample potentially containing one or more target molecules wherein each target molecule has a binding affinity for a specific affinity binding peptide tag; treating the sample with a fluorophore, wherein the fluorophore has a different excitation and emission peak than that of green fluorescent protein, under conditions effective to label the one or more target molecules with the fluorophore, if present in the sample; contacting the support with the sample under conditions effective to bind the one or more target molecules to the specific affinity binding peptide tags; and detecting a change in fluorescence of at least one of the green fluorescent protein molecules caused by binding of the one or more target molecules to the specific affinity binding peptide tags to identify the one or more target molecule.

64. The method according to claim 63, wherein the support is selected from the group consisting of beads, plastic supports, silicon supports, a protein chip, a multiwell device, glass supports, and membranes.

65. The method according to claim 63, wherein the green fluorescent protein is selected from the group consisting of a wild-type green fluorescent protein and variants thereof.

66. The method according to claim 65, wherein the variant green fluorescent protein is a mGFP5 variant, a GFP5-S65T variant, a cyan fluorescent protein variant, a yellow fluorescent protein variant, a red fluorescent protein variant, or a blue fluorescent protein variant.

67. The method according to claim 66, wherein the variant green fluorescent protein is the mGFP5 variant.

68. The method according to claim 66, wherein the variant green fluorescent protein is the GFP5-S65T variant.

69. The method according to claim 63, wherein each affinity binding peptide tag is selected from the group consisting of a metal-binding peptide, a carbohydrate-binding domain, a biotin-binding domain, an antigenic epitope, a nucleic acid-binding peptide, and a peptide that binds to a molecularly-imprinted polymer.

70. A microarray comprising a plurality of green fluorescent protein molecules immobilized at particular sites on a support, wherein each green fluorescent protein molecule comprises a distinctive affinity binding peptide tag inserted within a β- barrel structure of the green fluorescent protein.

71. The microarray according to claim 70, wherein the support is selected from the group consisting of beads, plastic supports, silicon supports, a protein chip, a multiwell device, glass supports, and membrane.

72. The method according to claim 70, wherein the green fluorescent protein is selected from the group consisting of a wild-type green fluorescent protein and variants thereof.

73. The method according to claim 72, wherein the variant green fluorescent protein is a mGFP5 variant, a GFP5-S65T variant, a cyan fluorescent protein variant, a yellow fluorescent protein variant, a red fluorescent protein variant, or a blue fluorescent protein variant.

74. The method according to claim 73, wherein the variant green fluorescent protein is the mGFP5 variant.

75. The method according to claim 73, wherein the variant green fluorescent protein is the GFP5-S65T variant.

76. The method according to claim 70, wherein each affinity binding peptide is selected from the group consisting of a metal-binding peptide, a carbohydrate- binding domain, a biotin-binding domain, an antigenic epitope, a nucleic acid-binding peptide, and a peptide that binds to a molecularly-imprinted polymer.