Field of the Invention This invention relates generally to the field of protein chemistry. Specifically, the present invention relates to protein purification using a metal ion affinity site.

Background of the Invention: Development of protocols for the isolation and purification of proteins is often a long and costly process. Such protocols usually contain multiple steps, where some of the steps have recoveries as low as 50%. Further, due to variation between protein molecules, a purification protocol developed and effective for the purification of one protein is not necessarily useful for the purification of another. In fact, in most cases considerable

adaptations must be made to a purification protocol to accommodate the various physical and chemical characteristics of different proteins.

The ability to prepare hybrid genes by genetic engineering technology has opened up new possibilities for the purification of proteins. For example, one can link a DNA sequence of a protein of interest to a nucleic acid sequence which codes for a peptide which has a high binding affinity for a specific ligand. The fusion protein product resulting from expression of this DNA has attributes of both the protein of interest and the high affinity peptide. To purify or immobilize the engineered fusion protein, the ligand commonly is linked to a support, and the unpurified, engineered protein is then exposed to the ligand/support composite and allowed to bind.

There are numerous advantages of using a high affinity fusion protein. For example, the use of an affinity peptide ensures that no part of the native protein of interest is involved in adsorption-the binding between the fusion protein and the ligand.

At the same time, extremely high selectivity in the adsorption process is achieved.

Immobilized Metal Ion Affinity Chromatography (IMAC) is one of the most frequently used techniques for purification of fusion proteins containing affinity sites for metal ions. Proper choice of immobilized metal ion, loading conditions and elution conditions can result in protein purification of up to about 95-98% in a single chromatographic step. Moreover, recovery generally is higher than 85%. In addition to the advantages discussed above, incorporation of a proteolytic, chemical, or enzymatic cleavage site into the composite DNA,

between the affinity peptide and the sequence of the protein of interest, provides a means for cleaving the affinity peptide from the protein of interest to yield the native protein of interest in highly purified form.

The following publications are representative of the art: Itakura, et al., Science 198: 1056-63 (1977); Germino, et al., PNAS USA 80: 6848-52 (1983); Nilsson et al., Nucleic Acid Res.

13: 1151-62 (1985); Smith et al., Gene 32: 321-27 (1984); Dobeli, et al., U. S. Pat. No. 5,284,933; and Dobeli, et al., U. S. Pat. No.

5,310,663.

The prior art is deficieint in improved compositions and methods for affinity immobilization and purification of proteins. This invention fulfills this long-felt need in the art.

SUMMARY OF THE INVENTION The present invention relates to compositions and methods for protein purification involving the use of novel, genetically-engineered fusion proteins. These fusion proteins are engineered to allow for immobilization and purification via th e high affinity interaction of an affinity peptide of a fusion protein with a ligand. The affinity peptide is a histidine-rich polypeptide sequence with a general sequence: (HXn) m, wherein H is histidine, X is an amino acid other than histidine, n= 1-8, m= 2-30, and wherein if n=1 for more than two adjacent units of HX, at least one X must be asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine. The affinity peptide

is linked to the proteins of interest Rl and R2 to yield a fusion protein with formula Rl- (HXn) m-R2. In a preferred embodiment of the invention, n=1-4 and m=2-10. In a more preferred embodiment of the invention, n=1-4 and m=3-6. In a specific embodiment of the invention, a fusion protein having the sequence SLKDHLIHNVHKEEHAHAHNKISVVGVGAVGM (SEQ ID No: 6) is provided. In another embodiment of the present aspect of the invention, at least one protease cleavage site is inserted between the sequence of the protein of interest and the sequence of the affinity peptide.

In another aspect of the invention, there is provided a DNA sequence coding for a fusion protein comprising a protein of interest fused at its amino-terminus or carboxy-terminus to a t least one affinity peptide, where the fusion protein has the general formula Rl- (HXn) m-R2, wherein RI or R2 is the protein of interest, H is histidine, X is an amino acid other than histidine, n= 1-8, m= 2-30, and wherein if n=1 for more than two adjacent units of HX, at least one X must be asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine. In a specific embodiment of this aspect of the invention, there is provided a DNA sequence which codes for a protein where the fusion protein has the sequence SLKDHLIHNVHKEEHAHAHNKISVVGVGAVGM (SEQ ID No: 6).

In various embodiments of this aspect of the invention, there is provided a recombinant vector comprising an expression vector and a DNA sequence coding for a fusion protein comprising a protein of interest fused at its amino-terminus or carboxy-terminus to at least one affinity peptide as described above, wherein the recombinant vector is capable of directing

expression of the DNA sequence in a suitable host organism. The present invention also provides a host organism containing a recombinant expression vector comprising a DNA sequence coding for a fusion protein comprising a protein of interest fused at its amino-terminus or carboxy-terminus to at least one affinity peptide as described above, wherein the organism is capable of expressing said DNA sequence.

In yet an additional aspect of this invention, there is provided a method for purifying the novel fusion proteins of the present invention, comprising the steps of: contacting a protein sample containing the fusion protein in a mixture with other proteins with a metal chelate resin under conditions where the fusion protein binds to the resin to produce a resin-fusion protein complex; washing the resin-fusion protein complex with a buffer to remove the other, unbound proteins; and eluting the bound fusion protein from the washed resin-fusion protein complex. One embodiment of this method includes inserting at least one protease cleavage site between the protein of interest and t h e affinity peptide, and cleaving the protein of interest from the affinity peptide after purification using the metal chelate resin.

Other and further aspects, embodiments, features and advantages of the present invention will be apparent from the following description of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic representation of the pGFPuv/HAT vector. This vector contains one embodiment of the

affinity peptide of the present invention fused to the N-terminus of a UV mutant of Green Fluorescent Protein. Only unique restriction sites are noted.

Figure 2 is a schematic representation of the vector pUC19/HS) containing part of the affinity peptide (AP) at the N- terminus of Enterokinase (EK) cleavage site followed by multiple cloning site (MCS). Only restriction sites are denoted.

Figure 3 is a schematic representation of the restriction maps of a vector with three frame shifts containing part of the affinity peptide and enterokinase cleavage site that is used for expression of recombinant proteins.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compositions and methods for purification of novel, genetically-engineered fusion proteins. Immobilization and purification is achieved via a high- affinity interaction of an affinity peptide portion of the fusion protein with a ligand. The high affinity peptide is a histidine-rich polypeptide with a general sequence (HXn) m which is linked to proteins of interest RI or R2; wherein H is histidine, Xis an amino acid other than histidine, n= 1-8, m= 2-30, and wherein if n=1 for more than two adjacent units of HX, at least one X must b e asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine. This high affinity polypeptide is incorporated by fusion to the N-or C-terminal sequence of a protein of interest and is used for high selectivity purification of the fusion protein.

The affinity of the high affinity peptide is for immobilized metal ions. The strength of binding between the high affinity peptide and an appropriate metal ion is very high; thus, isolation of the fusion proteins is very selective. However, association between the peptide and the ligand is also reversible.

Once the fusion protein has been allowed to associate or adsorb with the metal ion ligand, the protein can be disassociated or eluted from the metal ion/adsorbent by addition of competitive ligand such as imidazole, or by decreasing the pH, which leads to protonation of the nitrogen in the imidazole ring of the histidine side chain and release of the adsorbed protein. Because of this reversibility, the protein is recovered in a purified, unbound form.

Further, regeneration and reuse of the metal ion/adsorbent or support multiple times--even more than 100 times--is possible.

An additional feature of the protein purification and immobilization techniques based on the principles of the present invention is the high probability that the purified and regenerated protein of interest will retain full biological activity and specificity. This is because the affinity peptide is involved in the immobilization/binding process where the portion of the fusion protein that contains the protein of interest is not.

Incorporation of a proteolytic site between the high affinity peptide and the sequence of the protein of interest provides the means to regenerate the protein of interest from the fusion protein. Regeneration is achieved by limited proteolysis of the fusion protein and a second chromatography step in which the proteolytic product is passed through an immobilized metal ion affinity column. Indeed, one can utilize the same column as was used to immobilize and purify the fusion protein. In the second

chromatography step following proteolysis, the cleaved, regenerated protein of interest passes through the column without immobilization or adsorption, whereas the high affinity peptide is adsorbed on the column.

One embodiment of the present invention features incorporation of nucleic acid sequences which code for secretion signals into the DNA sequence that codes for the fusion protein.

Such secretion signals cause the fusion protein to be secreted into the media after synthesis in a host cell. Since a considerable amount of total cellular protein remains in the cell, secretion improves dramatically the isolation and purification of the fusion protein by eliminating the need for cell disruption, protein extraction, and/or removal of unwanted cellular components and nucleic acid.

As used herein, the terms"affinity peptide"or"high affinity peptide"refer to a histidine-rich polypeptide with a general sequence (HXn) m which is linked to proteins of interest RI or R2; wherein H is histidine, X is an amino acid other than histidine, n= 1-8, m= 2-30, and wherein if n=1 for more than two adjacent units of HX, at least one X must be asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine.

As used herein, the term"protein of interest"shall refer to any protein to which the affinity peptide is fused for the purpose of purification or immobilization.

As used herein, the term"fusion protein"shall refer t o the protein hybrid containing the affinity peptide and the protein of interest or any amino acid sequence of interest. The fusion protein has the general formula R1- (HXn) m-R2; wherein RI or R2 is

the protein of interest, H is histidine, X is an amino acid other than histidine, n= 1-8, m= 2-30, and wherein if n=l for more than two adjacent units of HX, at least one X must be asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine.

As used herein, the terms"secretion sequence"o r "secretion signal sequence"shall refer to an amino acid signal sequence which leads to the transport of a protein containing the signal sequence outside the cell membrane. In the present case, a fusion protein of the present invention may contain such a secretion sequence to enhance and simplify purification.

Representative examples of secretion signal sequences are well known to those having ordinary skill in this art.

As used herein, the term"proteolytic site"shall refer to any amino acid sequence recognized by any proteolytic enzyme.

In the present case, a fusion protein of the present invention may contain such a proteolytic site between the protein of interest and the affinity peptide and/or other amino acid sequences so that the protein of interest may be separated easily from these heterologous amino acid sequences.

As used herein, the term"metal ion"refers to any metal ion for which the affinity peptide has affinity and that can be used for purification or immobilization of a fusion protein.

Such metal ions include Ni+2, Co+2, Zn+2, Cu+2, Ac+3 and Fe+3.

As used herein, the terms"adsorbent"or"solid support"shall refer to a chromatography or immobilization medium used to immobilize a metal ion.

As used herein, the term"regeneration", in the context of the fusion protein, shall refer to the process of separating or

eliminating the affinity peptide and other heterologous amino acid sequences from the fusion protein to render the protein of interest after purification.

So that the matter in which the above-recited features, advantages, and objects of the invention become clear and can be understood in detail, particular descriptions of the invention may be had by reference to particular embodiments described in the Examples below; however, the following description and examples are given for the purpose of illustrating various, specific embodiments of the invention, and are not meant to limit the scope of the invention in any fashion.

EXAMPLE 1 Extraction of lactate dehvdrogenase from chicken breast muscle A naturally-occurring peptide sequence from the N- terminus of lactate dehydrogenase (LDH) from chicken muscle (Gallus gallus) was used for initial experiments. The protein includes a stretch of approximately 30 amino acids which has a sequence consistent with the general formula of the fusion protein of the present invention (SLKDHLIHNVHKEEHAHAHNKISVVGVGAVGM (SEQ ID No: 6)).

Further, LDH has the feature that the enzyme itself can be assayed easily for activity. Thus, the naturally-occurring chicken muscle LDH served as a"fusion protein"for these experiments in the sense that it contained both a high affinity peptide and a protein of interest.

Extraction of chicken breast muscle LDH was performed by cutting 15 g of frozen chicken muscle, free of blood vessels, into small pieces and transferring the material to a commercial blender along with 150 mL of extraction buffer (50 mM sodium phosphate, 1 mM EDTA, 1 mM magnesium acetate pH 7.5,1 mM 2-mercaptoethanol (0.2 L) stored for at least 3 0 minutes at 4°C). The mixture was homogenized twice at 4°C for 3 0 seconds, with a 10-minute pause between the bursts. After the second homogenization, the mixture was transferred to centrifuge tubes and centrifuged at 4°C and 10,000 x g for 30 minutes. The clear supernatant was collected and used as a starting sample for the purification of lactate dehydrogenase.

EXAMPLE 2 Purification of lactate dehydrogenase on Ni (II)-chelating sepharose FF Lactate dehydrogenase was purified by IMAC in the following manner: approximately 5 mL of Chelating Sepharose FF (Amersham, Pharmacia) was transferred to a vacuum bottle, diluted with an equal volume of deionized water and degassed under vacuum for 10 minutes. The gel suspension was poured into a column (10x1 cm. i. d.) trapped on the bottom with a degassed adapter and left to settle. The column was then filled to the top with degassed deionized water, and a top adapter was gently pushed down the column bed until there was no space between the top surface of the gel and the adapter. The column

was washed with 3 column volumes of deionized water at a flow rate of 0.5 mL per min.

The chicken muscle extract (14 mL) was equilibrated by gel filtration on Sephadex G-25 columns with equilibration buffer (20 mM sodium phosphate buffer containing 1.0 M sodium chloride and 0.06 M imidazole pH 7.0 (1 L)). The IMAC column was then charged with Ni (II) ions using 20 mL of a 0.02M Ni (NO3) 2 solution. The excess metal was washed from the column with deionized water at a flow rate of 0.5 mL per minute and the column was then equilibrated with 5-10 volumes of equilibration buffer (20 mM sodium phosphate buffer containing 1.0 M sodium chloride and 0.06 M imidazole pH 7.0 (1 L)).

The IMAC column was prepared by loading the equilibrated extract on to the IMAC column at a flow rate of 0.5 mL per min. Fractions of 1 mL were collected. The column was washed with equilibration buffer until a baseline was reached (absorbance of the fractions at 280 nm was less than 2 mAU higher than the absorbance of the equilibration buffer). The adsorbed material was eluted with elution buffer (20 mM sodium phosphate buffer containing 1.0 M sodium chloride and 0.3 M imidazole pH 7.0 (0.2 L)) and absorbance at 280 nm was determined on a spectrophotometer. Protein content of each fraction was determined as described in M. Bradford, Analytical Biochemistry, 72 (1976) 248, and lactate dehydrogenase activity was determined as described in F. Kubowitz and P. Ott, Biochem. Z, 314 (1943) 94. Results indicated that more than 95% of the lactate dehydrogenase activity was recovered in the elution fractions.

EXAMPLE 3 Characterization of lactate dehydrogenase binding Further experiments were performed both with native LDH, a tetramer of about 140 kD, and a subunit of the enzyme, obtained after warming of the crude chicken muscle extract to 45°C for 10 minutes. Both the tetramer and the subunit were allowed to associate with the immobilized Ni support, and both forms of LDH were retained. This result demonstrates that the retention of the LDH enzyme on immobilized Ni is not peculiar to the tetrameric form of the peptide; that is, binding does not require"cooperation"between subunits. Instead, the single subunit of the enzyme also had affinity for the nickel ion, and this affinity was demonstrated to be virtually identical to the affinity shown for the tetramer. Both the native protein and the subunit were adsorbed in buffer with an imidazole concentration up to 6 0 mmol and both were eluted completely at a concentration of 300 mmol imidazole.

To ascertain that it is the polyhistidine portion of the LDH that provides affinity for the nickel ion, the tetramic form of the LDH enzyme was subjected to CNBr cleavage to produce a mixture of peptides. This mixture of peptides was applied to a Ni- IDA column with metal ion capacity of 32 mmol per mL gel.

Loading conditions were the same as those used for the purification of the enzyme from the crude extract, described above. The adsorbed material was eluted with 300 mmol imidazole, and subjected to RPC chromatography. The chromatographic peak containing about 80% of the adsorbed

material was then subjected to amino acid analysis. The results obtained demonstrate that this peak corresponds to the N- terminal peptide from LDH and that this peptide that contains the polyhistidine sequence. In addition, the fact that the peptide retained its binding affinity even after treatment with CNBr in presence of 70% TFA is proof that the binding is not due to a rigid secondary conformation structure.

EXAMPLE 4 Purification of lactate dehydrogenase on Co2+-TALON agarose Extraction of chicken breast muscle LDH w a s performed as in Example 1, and equilibrated by gel filtration on Sephadex G-25 columns with equilibration buffer (20 mM sodium phosphate buffer containing 1.0 M sodium chloride and 0.06 M imidazole pH 7.0 (1 L)).

The IMAC column was prepared in the following manner: Approximately 2.75 mL of Co2+-TALON Superflow 6 (Amersham, Pharmacia) was transferred to a vacuum bottle, diluted with the same volume of deionized water and degassed under vacuum for 10 minutes. The gel suspension was poured into a column (3xl cm. i. d.) trapped on the bottom with a degassed adapter and left to settle. The column was filled to the top with degassed deionized water, and a top adapter was gently pushed down toward the column bed until there was no space between the top surface of the gel and the adapter. The column was washed with 3 column volumes of deionized water at a flow rate of 0.5 mL per min.

Purification of the fusion protein on Co2+-TALON Superflow 6 was carried out by first equilibrating the IMAC column with 5 to 10 column volumes of the equilibration buffer.

The sample was then loaded on the IMAC column at a flow rate of 1.0 mL per min, and 1 mL fractions were collected. The column was washed with the equilibration buffer until a baseline was reached (absorbance of the fractions at 280 nm as less than 2 mAU higher than the absorbance of the equilibration buffer).

The adsorbed material was eluted with elution buffer (20 mM sodium phosphate buffer containing 1.0 M sodium chloride and 0.3 M imidazole pH 7.0 (0.2 L)) and absorbance at 280 nm was determined on a spectrophotometer. Protein content of each fraction was determined as described in M. Bradford, Analytical Biochemistry, 72 (1976) 248, and lactate dehydrogenase activity was determined as described in F.

Kubowitz and P. Ott, Biochem. Z., 314 (1943) 94. As in Example 2, more than 95% of the lactate dehydrogenase activity was recovered in the elution fractions.

EXAMPLE 5 Isolation and purification of fusion protein consisting of affinity peptide and Green Fluorescent Protein UV Mutant (GFPuv) An affinity peptide/GFP fusion protein was isolated from E. coli cells which had been transformed with the pGFPuv. HS vector. Cell paste (0.39 g) was transferred to pre-cooled mortar, 1.2 g of alumina was added, and the mixture was ground for 2 minutes. Extraction buffer (5 mL, stored at 4°C) was added, and,

after additional grinding for 2 minutes, the mixture w a s transferred into four eppendorph tubes. The suspension was added to the eppendorph tubes and centrifuged for 12 minutes a t 12,000 rpm (11,750 x g). The clear supernatant (approximately 6 mL) was used as a starting sample for IMAC.

The extraction and chromatography equilibration buffers consisted of 20 mM sodium phosphate buffer containing 1.0 M sodium chloride and 5 mM imidazole pH 7.0 (1 L). The elution buffer for IMAC consisted of 20 mM sodium phosphate buffer containing 1.0 M sodium chloride and 150 mM imidazole pH 7.0 (0.2 L).

The IMAC was carried out in the following manner: Approximately 2.75 mL of Co2+-TALON Superflow 6 (Amersham, Pharmacia) was transferred to a vacuum bottle, diluted with the same volume of deionized water and degassed under vacuum for 10 minutes. The gel suspension was poured into a column (3xl cm. i. d.) trapped on the bottom with a degassed adapter and left to settle. The column was filled to the top with degassed deionized water, and a top adapter was gently pushed d o w n toward the column bed until there was no space between the top surface of the gel and the adapter. The column was washed with 3 column volumes of deionized water at a flow rate of 0.5 mL per min.

Purification of the fusion protein on Co2+-TALON Superflow 6 was carried out by first equilibrating the IMAC column with 5 to 10 column volumes of the equilibration buffer.

The sample was then loaded on the IMAC column at a flow rate of 1.0 mL per min, and 1 mL fractions were collected. The column was washed with the equilibration buffer until a baseline was

reached (absorbance of the fractions at 280 nm as less than 2 mAU higher than the absorbance of the equilibration buffer). The adsorbed material was then eluted with elution buffer.

Absorbance of each fraction at 280 nm w a s determined on a spectrophotometer; and protein content of each fraction was determined. Fluorescence of each fraction w a s determined on a microplate reader, and the purity of the fusion protein was determined also by SDS-electrophoresis. More than 85% of the fusion protein was recovered in the fractions obtained.

Part of the cDNA sequence, and the amino acid sequence encoded by this cDNA sequence, of a vector containing the affinity peptide at the N-terminus of Green Fluoresecent Protein-UV mutant (GFPuv) is shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

The full cDNA sequence of a vector containing the construct of the affinity peptide at the N-terminus of GFPuv is shown in SEQ ID No.

3. The full cDNA sequence of a vector containing part of the affinity peptide at the N-terminus of the enterokinase cleavage site and the amino acid sequence encoded by this cDNA corresponding to the start of translation site, the affinity peptide and the multiple cloning site are shown in SEQ ID Nos. 4 and 5.

EXAMPLE 6 Construction of fusion proteins A DNA sequence corresponding to the affinity peptide of the present invention is fused to the DNA coding sequence of a protein of interest. The polynucleotide sequence for the affinity peptide is fused most generally at or close to the DNA sequence

coding for the N-or C-terminal amino acid of the protein of interest. This results in a DNA sequence which codes for a fusion protein comprising the affinity peptide and the protein of interest.

In addition, a polynucleotide sequence that codes for a protein proteolytic site is incorporated into the fusion protein DNA sequence between the sequence for the affinity peptide and the sequence of the protein of interest. This type of DNA construct results in a fusion protein product having a proteolytic site. This site allows for the eventual regeneration of the protein of interest from the fusion protein by limited proteolysis and a second chromatography step. The second chromatography step, in which the product of the proteolysis is loaded onto an immobilized metal ion affinity column, results in the separation of the protein of interest from the affinity peptide.

An additional embodiment of the present invention provides a DNA sequence coding for a polypeptide"secretion signal"introduced into the DNA that codes for the fusion protein.

This secretion signal, when expressed, causes the fusion protein to be secreted into the culture media after the fusion protein is synthesized in the cell. Since a large number of cellular proteins are not transported out of the cell, isolation and purification of the fusion protein is enhanced as the requirements for cell disruption, extraction and removal of unwanted cell components are eliminated.

The present invention is directed to a fusion protein of general formula Rl- (HXn) m-R2 comprising: a protein of interest R1 or R2 fused at its amino terminus or carboxy terminus to at least one affinity peptide, said affinity peptide having a formula (HXn) m, wherein H is histidine, Xis an amino acid other than histidine, n=

1-8, m= 2-30, and wherein if n=l for more than two adjacent units of HX, at least one X must be asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine. Preferably, n=1-4 and m=3-10. In one preferred embodiment, n=1-4 and m=3-6. Preferably, if n=l for more than two adjacent units of HX, only one X is asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine. In one preferred embodiment, the fusion protein has a sequence SEQ ID No. 1. The fusion protein may contain at least one protease cleavage site between said protein of interest and said affinity peptide. Preferably, the affinity peptide has affinity for metal ions. A representative metal ion is a nickel ion. The fusion protein may further comprise a secretion signal sequence.

The present invention is also directed to a DNA sequence coding for a fusion protein of general formula R 1- (HXn) m-R2 comprising: a protein of interest R1 or R2 fused at its amino terminus or carboxy terminus to at least one affinity peptide, said affinity peptide having a formula (HXn) m, wherein H is histidine, X is an amino acid other than histidine, n= 1-8, m= 2- 30, and wherein if n=l for more than two adjacent units of HX, at least one X must be asparagine, phenylalanine, tryptophan, tyrosine, lysine, methionine, arginine, glutamine, or cysteine.

Preferably, the fusion protein has the sequence shown in SEQ ID No: 6.

The present invention is also directed to a recombinant vector comprising a DNA sequence disclosed herein, wherein said recombinant vector is capable of directing expression of said DNA sequence for said fusion protein in a suitable host organism. The present invention is also directed to a

host organism containing a recombinant vector disclosed herein, wherein said organism is capable of expressing said fusion protein.

The present invention is also directed to a method for purifying the fusion protein disclosed herein, comprising the steps of: contacting a protein sample containing said fusion protein in a mixture with other proteins with a metal chelate resin under conditions where said fusion protein binds to said resin to produce a resin-fusion protein complex; washing said resin-fusion protein complex with a buffer to remove said other, unbound proteins; and eluting said bound fusion protein from the washed resin- fusion protein complex; wherein said eluted fusion protein i s purified. This method may further comprise the step of cleaving said protein of interest from said affinity peptide. Moreover, this method further comprises the step of separating said cleaved protein of interest from said affinity peptide using a. metal chelate resin under conditions where said affinity peptide binds to said metal of said resin and said protein of interest does not.

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods,

procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.