WO2000018881A2

WO2000018881A2 - Intein mediated peptide ligation

Info

Publication number: WO2000018881A2
Application number: PCT/US1999/022776
Authority: WO
Inventors: Ming-Qun Xu; Thomas C. Evans
Original assignee: New England Biolabs, Inc.
Priority date: 1998-09-30
Filing date: 1999-09-30
Publication date: 2000-04-06
Also published as: CA2344764A1; DE69931382D1; JP2002526387A; DE69931382T2; CA2344764C; WO2000018881A8; EP1117693A2; WO2000018881A3; EP1117693A4; JP4749548B2; WO2000018881A9; EP1117693B1

Abstract

An in vitro method for producing a semi-synthetic fusion protein is provided, whereby a target protein fused to an intein - a protein splicing element - is selectively cleaved in a first step as depicted in Figure 1 with a thiol reagent, forming a carboxyl-terminal thioester of the target protein and releasing the target protein from the intein. In a subsequent step as shown in Figure 1, a desired, synthetic, protein or peptide having an amino-terminal cysteine is ligated to the target protein. Standard thiol-reagents such as DTT, or thiol-reagents optimized for ligation such as the odorless MESNA, may be used in the first step. The method permits the direct ligation of a desired peptide to a thioester bond that had linked a target protein to an intein. An in vivo variation of the method will permit production of a cytotoxic protein: a truncated, inactive, form of the protein fused to an intein is introduced in vivo, this fusion product is then selectively cleaved, and a synthetic protein or peptide is subsequently ligated at a carboxyl-terminal thioester of the target protein in order to restore the native activity of the cytotoxic protein.

Description

INTEIN MEDIATED PEPTIDE LIGATION

BACKGROUND OF THE INVENTION

Genetic engineering is a powerful approach to the manipulation of proteins. However, genetic methodologies are constrained by the use of only naturally coded amino acids. Furthermore, cytotoxic proteins are difficult to obtain by expression and isolation from a living source, since the expression of the toxic protein can result in death of the host.

To some extent, protocols have been developed to circumvent these problems, for example, total chemical synthesis (Kent, S. B. (1988) Ann. Rev. Biochem. 57:957-989), use of misacylated tRNAs (Noren, et al., (1989) Science

244:182-188), and semi-synthetic techniques (reviewed in Offord, R. (1987) Protein Eng. 1 :151 -157; Roy. et al. (1994) Methods in Enzymol. 231 :194-215; Wallace, C. J. (1993) FASEB 7:505-515). However, all of these procedures are limited by either the size of the fragment which can be generated or by low reaction yield.

It would therefore be desirable to develop a high-yield, semi-synthetic technique to allow in vitro fusion of a synthetic protein or peptide fragment to an expressed protein without limitation as to the size of the fused fragments. Likewise, in order to produce cytotoxic proteins, it would be desirable to develop a method of fusing a synthetic fragment, in vitro, to an inactive, expressed protein, so as to restore protein activity post-production from the host.

The modified See VMA intein has been used to generate thioester-tagged proteins for use in ligation (Example 19, U.S.S.N. 08/81 1 ,492, filed June 16, 1997; Chong, (1996) J. Biol. Chem., 271 (36):22159-22168; Chong, (1997) Gene,

192:271 -281 ; and Muir, et al. (1998) Proc. Natl. Acad. Sci USA 95:6705-6710).

Some disadvantages have been low yields due to poor cleavage of the See VMA intein with thiol-reagents that are optimum for ligation, the need for large peptide quantities due to on-column reactions, the use of odoriferous reagents, and/or low protein yields due to the use of a large, eukaryotic intein.

SUMMARY OF THE INVENTION

In accordance with the preseht invention, there is provided a method for producing a semi-synthetic fusion protein in vitro, comprising the steps of producing a target protein fused to a protein splicing element (an intein) and selectively cleaving the fusion and ligating a synthetic protein or peptide at the C-terminal thioester of the target protein, which overcome many of the disadvantages and problems noted above. Specifically, the present invention has higher yields due to better thiol-induced cleavage with thiol reagents which have been optimized for the ligation reaction.

Off-column ligation allows for sample concentration as well as the use of less peptide. In a particularly preferred embodiment, thiol reagents such as 2-mercaptoethanesulfonic acid (MESNA), which is an odorless thiol-reagent, is used for cleavage and ligation along with the Mxe intein, which is from a bacterial source and often expresses better in bacterial cells. Furthermore, the present invention allows peptides to be directly ligated to the thioester bond formed between an intein and the target protein. The present invention also provides a method for producing a cytotoxic protein, comprising the steps of producing a truncated, inactive form of the protein in vivo which is fused to a protein splicing element, and selectively cleaving the fusion and ligating a synthetic protein or peptide at a C-terminal thioester of the target protein to restore the activity of the native cytotoxic protein. Recombinant vectors for producing such cleavable fusion proteins are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow diagram depicting the chemical reactions which enable intein-mediated peptide ligation. The thioester generated at the C-terminus of the target protein during IMPACT™ purification was used in a 'native chemical ligation' reaction. This allowed the ligation of a synthetic peptide to a bacterially expressed protein. A typical ligation reaction involved the expression of the target protein-intein- CBD fusion followed by binding to a chitin resin. A thiol reagent induced cleavage of the intein. The target was eluted from the chitin resin and a synthetic peptide was added. The ligation reaction proceeded overnight.

Figure 2 is a gel depicting the results of cleavage and ligation reactions using various thiols. Cleavage and ligation reactions with different thiols visualized on 10-20% Tricine gels. MYB (a fusion protein of maltose binding protein-See VMA intein (N454A)-chitin binding domain) and MXB (a fusion protein of maltose binding protein-Mxe GyrA (N198A) intein- chitin binding domain) were incubated overnight at 4°C with various thiols (50 mM) in 150 mM Tris, 100 mM NaCl, pH 8 in the presence of a 30 amino acid peptide with an N-terminal cysteine. The peptide ligates to the C-terminus of MBP. Lanes

1 -5 ligation with MYB. Lane 1 no thiol. Lane 2 dithiothreitol. Lane 3 2-mercaptoethanesulfonic acid. Lane 4 3- mercaptopropionic acid. Lane 5 thiόphenol. Lanes 6-10 ligation with MXB. Lane 6 no thiol. Lane 7 dithiothreitol. Lane 8 2-mercaptoethanesulfonic acid. Lane 9 3-mercaptopropionic acid. Lane 10 thiophenol. Figure 3 is a gel depicting direct ligation of a peptide to the thioester formed between the See VMA intein and maltose binding protein. SDS-PAGE of direct ligation reaction with a 10-20% Tήcine gel. Lane 1 : a precursor protein (MYBIeu) consisting of maltose binding protein-See VMA1 intein-chitin binding domain was heated to >95°C for 5 minutes in a buffer of 50 mM Trizma base, pH 8.5 containing 100 mM NaCl, 1% SDS, and mM tris-(2-carboxyethyl)phosphine (TCEP) followed by overnight incubation at room temperature. The precursor (MYBIeu) is visible along with the See VMA1 intein (Y) and maltose binding protein (M), which are cleavage products. Lane 2: the precursor protein was subjected to the same conditions as described in Lane 1 except that the 30 amino acid peptide (1 mM) was added. The precursor (MYB) and cleavage products (Y and M) are visible along with the ligation product (M+30mer) formed when the 30 amino acid peptide fuses to maltose binding protein.

Figure 4 is a diagram depicting the pTXB1 expression vector of Example I (SEQ ID NO:7 and SEQ ID NO:8).

Figure 5 is the DNA sequence of pTXB1 (SEQ ID NO:5).

Figure 6 is a gel depicting the results of the Hpa\ protein ligation reaction. Protein ligation reactions examined on 10-

20% Tricine gels. Lane 1 : clarified cells extract after IPTG (0.5 mM) induction of ER2566 cells containing the pTXB2-Hpa/ plasmid. The fusion protein of Hpa/₂₂₃-Mxe GyrA-intein-CBD (52 kDa) is visible. Lane 2: cell extract as in Lane 1 after passage over a chitin column, which results in the binding of the fusion protein. Lane 3: Hpal₂₂₃ (25.7 kDa) after cleavage from the fusion protein by addition of MESNA. Lane 4: ligation product of Hpal₂₂₃ (0.2 mg/mL) with 1 mM of a 31 amino acid peptide (ligation product 29.6 kDa), representing the residues necessary to generate full length Hpal, after overnight incubation at 4°C. Lane 5: full length Hpal from a recombinant source (29.6 kDa) containing BSA (66 kDa) and two impurities.

Figure 7 is a western blot of various proteins ligated to a biotinylated peptide. Proteins purified with the Mxe GyrA IMPACT™ derivative were ligated to a synthetic peptide which contained an antibody recognition sequence.

DETAILED DESCRIPTION OF THE INVENTION

The ligation methods of the present invention are based on the discovery that a cysteine or peptide fragment containing an N-terminal cysteine may be fused, in vitro, to a bacterially expressed protein produced by thiol-induced cleavage of an intein (U.S. Patent No. 5,496,714; Example 19 of U.S.S.N. 08/81 1 ,492 filed June 16, 1997; Chong, et al., (1996) supra and Chong, et al., (1997) supra. The ligation procedure disclosed herein utilizes a protein splicing element, an intein (Perler, et al., (1994) Nucleic Acids Res. 22:1 125-1 127) to precisely create a thioester at the C-terminal -carbon of an expressed protein. - This reactive thioester could be present between the target protein and intein or generated by the addition of a thiol reagent. Previously the generation such a thioester was described using an intein (CIVPS) that was modified to undergo thiol inducible cleavage at its N-terminal junction in the presence of thiol reagent dithiothreitol (DTT) (Chong, et al. (1997) supra; Comb, et.al. U.S. Patent No. 5,496,714). This C-terminal thioester was previously used in a 'native chemical ligation' type reaction (Dawson, et al., (1994) Science 266:776-779) to fuse ³⁵S-cysteine or a peptide fragment containing an N-terminal cysteine to a bacterially expressed protein (Example 19, Comb, et.al. U.S. Patent No. 5,834,247, Chong (1996) supra and Chong (1997) supra.

The ligation method of the instant invention begins with the purification of the thioester-tagged target protein using an intein as described (Chong, et.al. (1997) supra). The direct ligation method of the instant invention begins with the isolation of a precursor composed of the target protein- intein-CBD. In one preferred embodiment, the host cell is bacterial. In other embodiments the host cell may be yeast, insect, or mammalian. A cysteine thiol at the N-terminus of a synthetic peptide nucleophilicly attacks a thioester present on the freshly isolated C-terminal α-carbon of the target protein or directly attacks the thioester present between the target protein and intein. This initially generates a thioester between the two reactants which spontaneously rearranges into a native peptide bond (Figure 1 ).

In order to optimize the ligation efficiency so that greater than 90% of the bacterially expressed target protein can be fused to the synthetic peptide or protein, specific thiol reagents and inteins are screened. In a preferred embodiment, the intein may be any CIVPS, such as See VMA, Mxe GyrA or derivatives of mutants thereof, and the thiol reagent is 2-mercapto-ethanesulfonic acid, thiophenol, DTT, or 3-mercaptopropionic acid (Comb, et al., U.S. Patent No. 5,496,714; U.S. Patent No. 5,834,247).

In one particularly preferred embodiment, an intein whose protein splicing activity has been blocked by mutation is utilized. The mutant must, however, retain the ability to undergo the N-S shift, thus allowing thioester formation between itself and an N-terminal protein. This thioester can then be nucleophilicly attacked by a thiol reagent or by the N- terminal cysteine of a peptide sequence. For example, by mutating the C-terminal asparagine (asn 198) of an intein from the GyrA gene of Mycobacterium xenopi (Telenti, et al.,

(1997) J Bacteriol 179:6378-6382) to an alanine created a thiol inducible cleavage element. This modified intein cleaved well with thiol reagents that were optimal for the ligation reaction, such as MESNA and thiophenol. Furthermore, optimal thiol reagent and intein combinations can be determined by incubating a precursor protein containing the intein of interest with a wide variety of thiol reagents followed by determination of the extent of cleavage of the precursor protein (Figure 2).

The use of such intein and specific thiol reagents leads to optimal yields and high ligation efficiencies; typically greater than 90% of the N-terminal ligation fragment can be modified.

The ligation methods of the present invention expand the ability to incorporate non-coded amino acids into large protein sequences by generating a synthetic peptide fragment with fluorescent probes, spin labels, affinity tags, radiolabels, or antigenic determinants and ligating this to an in vivo expresed protein isolated using a modified intein.

Furthermore, this procedure allows the isolation of cytotoxic proteins by purifying an inactive truncated precursor from a host source, for example bacteria, and generating an active protein or enzyme after the ligation of a synthetic peptide. For example, restriction endonucleases which have not successfully been cloned by traditional methods may be produced in accordance with the present invention.

Also, the direct ligation procedure allows the ligation of a protein or peptide sequence to another protein or peptide sequence without the use of exogenous thiol reagents. Direct ligation relies on the nucleophilic attack of the N-terminal amino acid of one peptide on the thioester formed between a target protein and an intein (Figure 3).

In summary, a fusion protein can be created using the methods of the present invention that possesses unique properties which, currently, can not be generated genetically.

The Examples presented below are only intended as specific preferred embodiments of the present invention and are not intended to limit the scope of the invention. The present invention encompasses modifications and variations of the methods taught herein which would be obvious to one o f ordinary skill in the art.

The references cited above and below are herein incorporated by reference. EXAMPLE I

Creation of vectors pTXB1 and pTXB2 for ligation:

Asparagine 198 of the Mxe GyrA intein (Telenti, et al.,

(1997) J Bacteriol. 179:6378-6382) was mutated to alanine by linker insertion into the Xmn\ and Pst\ sites of pmxeMIPTyrXmnSPdel to create pMXP1. The Xmn\ site was originally introduced into the unmodified Mxe GyrA intein sequence by silent mutagenesis. The Pst\ site was a unique site in the plasmid. The linker was composed of mxe#3 (5¹- GGTTCGTCAGCCACGCTACTGGCCTCACCGGTTGATAGCTGCA-3') (SEQ ID NO:1) and mxe#4 (5'-GCTATCAACCGGTGAGGCCAGTAG CGTGGCTGACGAACC-3') (SEQ ID NO:2).

Into pMXP1 another linker composed of mxe#1 (5'-TC GAATCTAGACATATGGCCATGGGTGGCGGCCGCCTCGAGGGCTCTTCC TGCATCACGGGAGATGCA-3') (SEQ ID NO:3) and mxe#2 (5'-CTAG TGCATCTCCCGTGATGCAGGAAGAGCCCTCGAGGCGHGCCGCCACCCA TGGCCATATGTCTAGAT-3') (SEQ ID NO:4) was inserted into the

Xho\ and Spel sites to introduce a multiple cloning site (Xba\- Nde\-Nco\-Not\-Xho\-Sap\) before the Mxe GyrA intein (pMXP2).

The 0.6 kilobase Not\ to Age\ fragment of pMXP2 was ligated into the same sites in pTYB1 (IMPACT kit, New England

Biolabs, Beverly, MA) and the Λ/col to Age\ fragment of pMXP2 was cloned into pTYB3 (IMPACT kit, New England Biolabs, Beverly, MA) to create plasmids pTXB1 (see Figure 4 and 5) (SEQ ID NO:5) and pTXB2, respectively. These vectors have a multiple cloning site upstream of the modified Mxe GyrA intein-chitin binding domain fusion. This allows the insertion of a target gene of interest inframe with the intein and chitin binding domain (CBD).

Creation of vectors pMYBieu for ligation:

pMYBIeu was as described in Chong, et al., (1998), J. Biol. Chem. 273:10567-10577. This vector consisted of maltose binding protein upstream of the See VMA intein-chitin binding domain. A leucine is present at the -1 position instead of the native residue (which is a glycine).

Purification of Thioester-Tagged Proteins:

Protein purification was as described using the See VMA intein (Chong, et.al., (1997) Gene 192:271 -281 ) with slight modification. ER2566 cells (IMPACT T7 instruction manual from New England Biolabs, Beverly, MA) containing the pTXB vector with the appropriate insert were grown to an OD600 of 0.5-0.6 at 37°C at which point they were induced with 0.5 mM IPTG overnight at 15°C. Cells were harvested by centrifugation and lysed by sonication (performed on ice). The three part fusion protein was bound to chitin beads (10 mL bed volume, Figure 6, lanes 1 and 2) equilibrated in Buffer A (50 mM Tris, pH 7.4, and 500 mM NaCl), and washed with 10 column volumes of Buffer A to remove unbound material.

Cleavage was initiated using a buffer of 50 mM 2- mercaptoethanesulfonic acid (MESNA), 50 mM Tris, pH 8.0 and 100 mM NaCl. Other thiol reagents were also used at other times, such as thiophenol, dithiothreitol, and/or 3- mercaptopropionic acid. After overnight incubation at from 4-

25°C protein was eluted from the column (Figure 6 lane 3). This protein contained a thioester at the C-terminus.

Purification of MYB. MYBIeu and MXB:

Full length precursor proteins consisting of maltose binding protein-See VMA intein (N454A)-chitin binding domain (MYB) and maltose binding protein-Mxe GyrA (N198A) intein- chitin binding domain (MXB) were purified after induction and sonication, as described above, by applying the sonicated sample to a 10 mL column of amylose resin (New England Biolabs, Beverly, MA). Unbound proteins were washed from the column with 10 column volumes of Buffer A (see purification of thioester-tagged proteins). Bound proteins were eluted with a buffer of 50 mM Tris, pH 8, containing 100 mM NaCl and

10 mM maltose. Fractions were collected and protein concentrations were determined using the Bio-Rad Protein Assay (Hercules, CA).

Peptide Synthesis:

Peptides for subsequent ligation reactions were synthesized on an ABI model 433A peptide synthesizer utilizing FastMoc™ chemistry (Fields, et al., (1991 ) Pept Res 4, 95-101 ) at a 0.085 mmol scale. Preloaded HMP (p- hydroxymethylphenoxymethyl) polystyrene resins (Applied

Biosystems, Foster City, CA) functionalized at 0.5 mmol/g was used in conjunction with Fmoc/NMP chemistry utilizing HBTU amino acid activation (Dourtoglou, et al., (1984) Synthesis 572-57 '4; Knorr, et al., (1989) Tetrahedron Lett 30, 1927-1930). Fmoc amino acids were purchased from Applied

Biosystems (Foster City, CA).

Synthesis proceeded with a single coupling during each cycle. Peptide cleavage from the resin and simultaneous removal of side chain protecting groups was facilitated by the addition of cleavage mixture (Perkin Elmer, Norwalk, CT) consisting of 0.75 g phenol, 0.25 mL 1 ,2-ethanedithiol, 0.5 mL deionized H2O, and 10 mL TFA. The resin was flushed with nitrogen and gently stirred at room temperature for 3 hours. Following filtration and precipitation into cold (0°C) methyl- t-butyl ether, the precipitate in the ether fraction was collected by centrifugation. The peptide precipitate was vacuum dried and analyzed by mass spectrometry using a Perceptive Biosystems (Framingham, MA) MALDI-TOF mass spectrometer.

Final purification was by HPLC using a Waters HPLC system with a Lambda-Max Model 481 Multiwavelength detector (set at 214 nm), 500 series pumps and automated gradient controller with a Vydac semi-preparative C18 column. Elution of the peptide was with a 60 minute linear gradient of 6-60% acetonitrile (v/v) in an aqueous solution of 0.1 % TFA (v/v).

Protein Cleavage and Ligation Reactions:

Cleavage of MYB and MXB: The precursor protein (1 mg/mL) was incubated overnight at 4°C with or without a thiol reagent (50 mM) in 150 mM Tris, pH 8, containing 100 mM NaCl.

Ligation reactions with MYB and MXB: The precursor protein (1 mg/mL) was treated as described for cleavage except that a 30 amino acid peptide (1 mM final concentration, NH2-CAYKTTQANKHIIVACEGNPYVPVHFDASV-COOH (SEQ ID NO:6) was also included in the reaction (Figure 2). Ligation reactions after purification of thioester-tagged proteins: Lyophilized peptides (New England Biolabs, Beverly, MA) were added (to 1 mM final concentration) directly to the thioester-tagged protein freshly isolated from the chitin column. The reaction was allowed to proceed overnight at from 4-25°C. In both ligation procedures the condensation of the reactants is visible on a 10-20% Tricine gel (Figure 6). The ligation reaction was tested in conditions of 5-150 mM Tris or HEPES buffers, 50-1000 mM NaCl, 10 mM Maltose, and pH 6-11 and 0-6 M Urea.

Direct Ligation Reactions:

MYBIeu (1 mg/mL) was incubated in 6 M Urea or 1 % SDS, pH 7.5-8.5, 50-200 mM NaCl, and 1 mM of a 30 amino acid peptide (NH₂CAYKTTQANKHIVVACEGNPYVPVHFDASV-COOH (SEQ ID NO:6)). The MYBIeu was incubated for 0-180 minutes at either 4°C or 100°C prior to the addition of the 30 amino acid peptide. Ligation reactions proceeded overnight at either 4°C or 25°C.

EXAMPLE II

Labeling a target protein: Maltose Binding Protein

Maltose binding protein (MBP, 42 kDa) was isolated as described in Example I above using the IMPACT procedure (IMPACT manual from New England Biolabs, Inc., Beverly, MA) in the presence of MESNA.

A biotinylated peptide possessing an N-terminal cysteine (CDPEK^*DS-COOH (SEQ ID NO:9)), in which the biotin was attached to the ε-amino group of the lysine residue) was ligated to the freshly purified target protein as described above. Briefly, 4 μL of biotinylated peptide (10 mM) were mixed with a 36 μL aliquot of the freshly purified MBP sample.

The mixture was incubated at 4°C overnight.

Western blots with alkaline phosphatase linked anti- biotin antibody detected the presence of the ligated product but not the unligated target protein (Figure 7). The efficiency of the ligation is typically greater than 90% when MESNA is used for cleavage. EXAMPLE

Labeling a target protein: Bst DNA Polymerase I Large Fragment (Bst Pol 1)

Bst DNA Polymerase I large fragment (67 kDa) was isolated as described in Example I above using the IMPACT procedure (IMPACT manual from New England Biolabs, Inc., Beverly, MA) in the presence of MESNA.

A biotinylated peptide possessing an N-terminal cysteine (CDPEK^*DS-COOH (SEQ ID NO:9)), in which the biotin was attached to the ε-amino group of the lysine residue) was ligated to the freshly purified target protein as described. Briefly, 4 μL of biotinylated peptide (10 mM) were mixed with a 36 μL aliquot of the freshly purified Bst Pol 1 sample. The mixture was incubated at 4°C overnight.

Western blots with alkaline phosphatase linked anti- biotin antibody detected the presence of the ligated product but not the unligated target protein (Figure 7). The efficiency of the ligation is typically greater than 90% when MESNA is used for cleavage. EXAMPLE IV

Labeling a target protein: Paramyosin

Paramyosin (29 kDa) was isolated as described in

Example I above using the IMPACT procedure (IMPACT manual from New England Biolabs, Inc., Beverly, MA) in the presence of MESNA.

A biotinylated peptide possessing an N-terminal cysteine (CDPEK*DS-COOH (SEQ ID NO:9)), in which the biotin was attached to the ε-amino group of the lysine residue) was ligated to the freshly purified target protein as described. Briefly, 4 μL of biotinylated peptide (10 mM) were mixed with a 36 μL aliquot of the freshly purified paramyosin sample. The mixture was incubated at 4°C overnight.

Western blots with alkaline phosphatase linked anti- biotin antibody detected the presence of the ligated product but not the unligated target protein (Figure 7). The efficiency of the ligation is typically greater than 90% when MESNA is used for cleavage. EXAMPLE V

Labeling a target protein: E. coli Thioredoxin

E. coli thioredoxin (12 kDa) was isolated as described in

A biotinylated peptide possessing an N-terminal cysteine (CDPEK^*DS-COOH (SEQ ID N0.9)), in which the biotin was attached to the ε-amino group of the lysine residue) was ligated to the freshly purified target protein as described. Briefly, 4 μL of biotinylated peptide (10 mM) were mixed with a 36 μL aliquot of the freshly purified thioredoxin sample. The mixture was incubated at 4°C overnight.

Western blots with alkaline phosphatase linked anti- biotin antibody detected the presence of the ligated product but not the unligated target protein (Figure 7). The efficiency of the ligation is typically greater than 90% when MESNA is used for cleavage. EXAMPLE VI

Isolation of a cytotoxic protein:

The ligation procedure of Example I was applied to the isolation of a potentially cytotoxic protein. An endonuclease from Haemophilus parainfluenzae (Hpal, Ito, et al., (1992) Nucleic Acids Res 20:705-709) was generated by ligating an inactive truncated form of the enzyme expressed in E. coli (ER2566 cells, New England Biolabs, Inc., Beverly, MA) with the missing amino acids that were synthesized chemically.

The first 223 amino acids of Hpal (full length Hpal is 254 amino acids) were fused in frame with the modified Mxe GyrA intein and the CBD. The 223 amino acid Hpal fragment was isolated as described for purification of thioester tagged proteins. The truncated Hpal displayed no detectable enzymatic activity.

A synthetic peptide representing the 31 amino acids needed to complete Hpal was ligated onto the 223 amino acid truncated form of Hpal by the method of Example I. Enzymatic Assay for Hpal :

The activity of the fused Hpal was determined by its ability to digest Lambda DNA (New England Biolabs, Beverly, MA). Serial dilutions of ligated or truncated Hpal, with the appropriate peptide added to 1 mM, were incubated with 1 μg of Lambda DNA for 1 hour at 37°C in a buffer of 20 mM Tris- acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, and 170 μg/mL BSA (total volume 30 μL). Digestion reactions were visualized on 1% agarose gels permeated with ethidium bromide. One unit of Hpa I was defined as the amount of enzyme necessary to digest 1 μg of Lambda DNA in one hour at 37°C.

The newly ligated Hpal had a specific activity of 0.5-

1 .5x10⁶ units/mg which correlated well with the expected value of 1 -2x10⁶ units/mg for the full length enzyme.

Claims

WHAT IS CLAIMED IS:

1 . A method for fusing an expressed protein with a peptide, said method comprising the steps of:

(a) generating at least one C-terminal thioester- tagged target protein;

(b) generating at least one target peptide having a specified N-terminal; and

(c) ligating said target peptide to said target protein.

2. The method of claim 1 , wherein said target protein is generated from a first plasmid comprising an intein having N-terminal cleavage activity.

3. The method of claim 2, wherein said intein comprises an intein having a cysteine residue at the N-terminal of the intein.

4. The method of claim 3, wherein said target protein is generated by thiol reagent-induced cleavage of said intein.

5. The method of claim 4, wherein said thiol reagent is selected from the group consisting of MESNA, thiophenol, DTT, β-mercaptoethanol or derivatives thereof.

6. A fusion protein produced by the method of any one of claims 1 -5.

7. A cyclic protein produced by the method of claim 1.

8. A modified intein comprising a mutant Mxe GyrA intein capable of thiol reagent-induced cleavage to produce a thioester at the C-terminal of an adjacent target protein.

9. A method of generating a reactive thioester comprising contacting a thiol reagent selected from the group consisting essentially of MESNA, thiophenol, DTT, β- mercaptoethanol or derivatives thereof with a precursor comprising a target protein and intein.

10. A method for screening thiol reagents which cleave a target intein comprising the steps of:

(a) isolating a precursor comprising a protein and a modified intein;

(b) contacting a thiol reagent with the precursor of step (a);

(c) determining whether a splicing or cleaving event occurs.

1 1 . The method of claim 10, comprising the further step of determining whether the spliced or cleaved product of step (c) can ligate to a target peptide having an N- temrinal cytokine.