KR20030097036A - Recombinant enterokinase by modifying N-terminus or C-terminus of enterokinase light chain - Google Patents

Abstract

PURPOSE: A recombinant enterokinase of which N-terminal or C-terminal of light chain is modified is provided, thereby easily recovering and removing the enterokinase after its reaction, and easily mass-producing the enterokinase in Escherichia coli. CONSTITUTION: A recombinant enterokinase is characterized by having modified N-terminal or C-terminal of light chain, wherein the modification is carried out by adding an affinity tag to the carboxy-terminal of enterokinase light chain; the affinity tag is selected from the group consisting of histidine(His) tag, haemagglutinin(HA) tag, poly arginine tag, poly lysine tag, flag tag and myc tag; the recombinant enterokinase protein comprises the amino acid sequence of SEQ ID NO: 9; the modification is carried out by substituting isoleucine of amino terminal of enterokinase light chain by valine; and the recombinant enterokinase comprises the amino acid sequence of SEQ ID NO: 17.

Description

Recombinant enterokinase by modifying N-terminus or C-terminus of enterokinase light chain}

The present invention relates to a recombinant enterokinase protein wherein the amino or carboxyl terminus of an enterokinase light chain protein is modified, and more specifically, an affinity tag is attached to the carboxyl terminus of an enterokinase light chain. A recombinant enterokinase protein inserted with or substituted with an amino-terminal isoleucine with valine, a gene thereof, an expression vector containing the gene, a transformant transformed with the expression vector, and the target protein using the recombinant enterokinase protein It relates to a manufacturing method.

With the development of genetic recombination technology, a lot of useful foreign proteins using prokaryotes such as animal cells, yeast or E. coli have been produced, and these proteins are widely used in the biotechnology industry as medicines. In particular, E. coli has a rapid growth rate of cells and its genes have been well studied compared to other organisms, and thus are widely used as host cells for producing foreign proteins in gene recombination technology. Escherichia coli, however, has a critical disadvantage in that it does not have the various intracellular elements necessary for the maturation of proteins in eukaryotes. Specifically, E. coli does not perform functions such as post-translational modification, disulfide bond formation, glycosylation and compartmentalization of eukaryotes. In addition, when foreign proteins are excessively expressed in Escherichia coli, these proteins are often accumulated in the cytoplasm in the form of an inclusion body (insoluble aggregate). Therefore, in order to improve the above problems caused when the foreign protein is produced in aggregate form, it is important to express the foreign protein in water-soluble protein form in E. coli. The most effective in water soluble expression of foreign proteins in E. coli is fusion protein technology. This technique has increased in post-genomics in the functional genomics and structural genomics of many proteins. Enzymes such as enterokinase, thrombin, and factor Xa, which specifically cleave specific protein sequences, are essential for obtaining the target protein in the fusion protein. However, these cleavage enzymes are expensive, and despite the many advantages of the fusion protein technology, it is the biggest obstacle to the industrial use. Therefore, the development of improved performance of recombinant enterokinase is expected to contribute significantly to the fusion protein technology.

Enterokinase cleaves the amino acid sequence Val- (Asp) ₄ -Lys, described as SEQ ID NO: 1 , the amino terminal peptide of trypsinogen, converts it to trypsin and activates many pancreatic zymogens (Kunitz , J. Gen. physiol ., 1939, 22: 429-446; Yamshina, Acta Chem. Scand ., 1956, 10: 739: 743). The amino acid sequence (Asp) ₄ -Lys, set forth in SEQ ID NO: 2 , is a site conserved as a recognition site for enterokinase in many vertebrates, which inhibits auto-activation of trypsinogen by trypsin (Maroux et al., J. Biol. Chem ., 1971, 246: 5031-39). Thus, enterokinase has been widely used for the location-specific cleavage of fusion proteins with high specificity for the amino acid sequence set forth in SEQ ID NO: 2 (LaVallie et al., J. Biol. Chem. , 1993, 268: 23311-17).

Small enterokinase is a heterodimer that is a double-sulfur bond consisting of a 35 kDa light chain and a 115 kD heavy chain derived from a single stranded precursor (Kitamoto et al., Proc. Natl. Acad, Sci. USA , 1994, 91: 7588-92). Among them, enterokinase light chain (abbreviated as "EK _L ") is secreted by partial reduction of the homoenzyme and shows protein resolution (Light and Fonseca, J. Biol. Chem. , 1984, 259: 13195). -98), the heavy chain is involved in trypsinogen recognition and membrane binding (Fonseca and Light, J. Biol. Chem ., 1983, 258: 14516-20). Duodenase is believed to play a role in activating pro-enterokinase (Zamolodchikaba et al., FEBS Lett ., 2000, 466: 295-299). CDNA encoding bovine EK _L was isolated and isolated from mammalian COS cells (LaVallie et al., J. Biol. Chem . 1993, 268: 23311-17), E. coli using DsbA as a fusion partner (Collins-Racie et al., Bio / technology , 1995, 13: 982-987) and the results of expression of the host in the host such as methylotropic enzyme Pichia pastoris (Vozza et al., Bio / technology, 1996, 14: 77-81) Is being reported.

Since the amino terminus of enterokinase is isoleucine, the production of active protein is very difficult because methionine is not removed by methionine aminopeptidase when produced directly in E. coli.

Although the recognition site for enterokinase is known to be very specific, the minimum sequence for cleavage is the acidic amino acid of the P2 position ((Asp) ₄ -Lys at the last Lys is P1 and conversely the first Asp is at the P5 position). Glutamic acid or aspartic acid) and basic amino acids (lysine or arginine) at the P1 position. In addition, the rate of hydrolysis increases as the acidic residues located at the P3-P5 site increase.

Since enterokinase remains in the reaction solution even after cleaving the fusion protein, the target protein can be decomposed. Therefore, in order to prevent degradation of the target protein, it is preferable to remove the enterokinase after the fusion protein is cleaved. However, there is no conventional method for removing enterokinase inexpensively, so there are many difficulties in separating target proteins.

Accordingly, the present inventors know that enterokinase degrades the target protein and degrades the final product yield during cleavage of the fusion protein. Alternatively, the recombinant enterokinase prepared at the carboxyl terminal may be prepared, and the recombinant enterokinase may be easily purified. The recombinant enterokinase may be easily purified after cleavage of the fusion protein, thereby increasing the yield of a desired protein. The present invention was completed by mass production by refolding in Escherichia coli by performing systematic mutation of amino terminus (mutagenesis).

An object of the present invention is a recombinant enterokinase with improved enzyme function through modification of amino and carboxyl ends of a light chain of recombinant enterokinase, and the separation, cleavage and recovery of proteins using the recombinant enterokinase, and the like. It is to provide a way to easily obtain the target protein in the fusion protein technology.

1 is a schematic diagram of a vector pIL20EK _L having a bovine enterokinase light chain cDNA inserted at the restriction enzymes XbaI and BamHI positions of the expression vector pIL20GH,

Ap: ampicillin resistance gene, 2μ: replication origin of 2μ plasmid,

GAL4: galactose-induced gene positive regulatory protein in yeast,

URA3: gene encoding orotidine-5-phosphate decarboxylase,

pMFα1: promoter of yeast mating factor α1,

UASg: GAL1-10 higher active sequence, ktl: killer toxin leader sequence,

tGAPDH: transcriptional terminator of glyceraldehyde phosphate dehydrogenase,

FIG. 2 is an electrophoresis photograph analyzed by SDS-PAGE of recombinant EK _L purified by benzamidine sepharose column chromatography with endoglycosidase H.

Lane 1: size marker,

Lane 2: recombinant EK _L purified by benzamidine sepharose column chromatography _,

Lane 3: recombinant EK _L treated with endoglycosidase H _,

Figure 3a is an electrophoresis picture of SDS-PAGE analysis of cleavage reaction of fusion protein T2GH using recombinant EK _L ,

Lane 1: size marker, lane 2 and lane 5: fusion protein T2GH,

Lane 3: reaction for 2 hours with recombinant EK _L , lane 4: reaction for 4 hours with recombinant EK _L ,

Lane 6: reaction for 8 hours with recombinant EK _L ,

Figure 3b is an electrophoresis picture of SDS-PAGE analysis of cleavage reaction of the fusion protein T2GH with yeast culture supernatant of bovine enterokinase and other recombinant enterokinase,

Lane 1: size marker, lane 2: fusion protein,

Lane 3: treatment with bovine enterokinase at 0.5 μg,

Lane 4: treated with 0.5 μg bovine enterokinase and 10 μl medium culture,

Lane 5: treatment with the culture supernatant of the transformants into which the pIL20X vector was introduced,

Lane 6: treatment with the culture supernatant of the transformants into which the pIL20XΔEK _L vector was introduced,

Lane 7: treatment with the culture supernatant of the transformant into which the pIL20XGH vector was introduced,

Lane 8: treatment with the culture supernatant of the transformants into which the pIL20XEK _L vector was introduced,

4 is an electrophoresis photograph of SDS-PAGE analysis of purified EK _L -Ca-His at once by nickel affinity chromatography;

Lane 1: size marker,

Lane 2: extraction from the column washed with buffer B,

Lane 3: purified recombinant EK _L -Ca-His,

5 is a graph comparing enterokinase activity of EK _L , recombinant EK _L -Ca-His and recombinant N-His-EK _L using a fluorescing substrate,

△: EK _L , O: recombinant EK _L -Ca-His, □: recombinant N-His-EK _L ,

Figure 6 is a schematic diagram of a mutant expression vector mutated amino terminal of the enterokinase light chain,

7 is a graph comparing the enzyme activity of the I1V mutation and wild type enterokinase,

■: I1V mutation, ▲: wild type enterokinase (EK _L ),

△: human growth hormone (hGH) (control)

8 is a graph measuring the enzyme activity according to the temperature of the I1V mutation and wild type enterokinase,

A: 45 ° C, B: 65 ° C,

■: I1V mutation, ▲: wild type enterokinase (EK _L ),

9 is a graph comparing removal of EK _L and recombinant EK _L -Ca-His with a nickel NTA spin column.

Δ: EK _L , O: recombinant EK _L -Ca-His

In order to achieve the above object, the present invention provides a recombinant enterokinase protein modified amino or carboxyl terminal of the enterokinase light chain and a gene encoding the same.

The present invention also provides an expression vector comprising the gene encoding the recombinant enterokinase and transformants transformed with the expression vector.

The present invention also provides a method for preparing a protein of interest by cleaving a fusion protein using the recombinant enterokinase protein and recovering the recombinant enterokinase protein in the reaction solution to separate the protein of interest.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant enterokinase protein modified at the amino or carboxyl terminus of an enterokinase light chain and a gene encoding the same.

The recombinant enterokinase protein of the present invention may add an affinity tag to the carboxyl terminus of the enterokinase light chain, and the affinity tag may be a histidine tag, a hemagglutinin (HA) tag, a poly arginine tag, a poly Lysine (poly Lysine) tag, flag (flag) tag and myc tag and the like can be used, and not necessarily limited to the affinity tag, affinity tag that can be easily separated and purified can be used. The recombinant enterokinase protein of the present invention can be easily recovered or removed in the reaction solution by a known separation method using the affinity tag, or can be immobilized using an affinity tag.

The present invention also provides a recombinant enterokinase protein wherein the recombinant enterokinase protein is substituted with valine for the amino-terminal isoleucine of the enterokinase light chain. As described above, the recombinant enterokinase protein substituted with valine for the amino-terminal isoleucine is expressed in E. coli, which can be mass-produced, and thus industrially useful.

The recombinant enterokinase protein of the present invention is preferably a recombinant enterokinase protein in which the carboxyl terminus of the light chain of the enterokinase is added with an affinity tag and the amino terminus isoleucine is substituted.

In the recombinant enterokinase protein of the present invention, the affinity tag may be located at the amino terminus or the carboxyl terminus, but more preferably at the carboxyl terminus. In addition, the number of affinity tags can be easily changed, for example the number of histidines in His-tags can be varied to suit the application. In addition, a spacer can be inserted between the carboxyl terminus of the light chain of the enterokinase and the affinity tag. For example, inserting a glycine-serine-glycine-serine-glycine spacer in the middle does not change the activity of the enzyme.

In a preferred embodiment of the invention, the recombinant enterokinase protein His-tagged at the carboxyl terminus of the enterokinase light chain has an amino acid sequence as set out in SEQ ID NO: 9 , and the gene encoding the base sequence as set forth in SEQ ID NO: 10 Have However, it is natural for those skilled in the art that all base sequences capable of producing the amino acid sequence when included in addition to the base sequences are included in the scope of the present invention. Such nucleotide sequences may be prepared by substitution, deletion or insertion of the nucleotide sequences set forth in SEQ ID NO: 10 .

In a preferred embodiment of the present invention, the recombinant enterokinase protein substituted with valine for the amino-terminal isoleucine of the enterokinase light chain has an amino acid sequence represented by SEQ ID NO: 17 , and the gene encoding the same is represented by SEQ ID NO: 18 . Has a nucleotide sequence

In a preferred embodiment of the invention, the activity of the rEK _L- Ca-His of the present invention His-tagged at the carboxyl terminus increased with time similar to recombinant EK _L , but His-tagged at the carboxyl terminus -N-rEK _L lacks EK _L activity (see FIG. 5 ). This means that the nature of the amino terminal residues of the recombinant EK _L rather than the carboxyl terminal plays an important role in enterokinase activity. This is consistent with this technique in which the amino-terminal amino acid isoleucine of recombinant EK _L is involved in salt bridges that are crucial in structure and thus amino-terminal extension inhibits enzyme activity (Fehlhammer et al., J. Mol. Biol ., 1977, 11: 415-438; Collins-Racie et al., Bio / technology , 1995, 13: 982-987). In addition, the three-dimensional structure of the recombinant EK _L shows that the amino-terminal isoleucine residues are located inside the protein for the salt bridge, while the carboxyl-terminal sites are external (Lu et al., J. Mol., Biol). , 1999, 292: 361-373).

The present invention also provides an expression vector comprising the gene encoding the recombinant enterokinase protein and a transformant transformed with the expression vector.

The present invention includes a gene encoding a recombinant enterokinase protein, and provides an expression vector capable of mass production of the recombinant enterokinase protein.

We have previously produced a new separation system in recombinant Saccharomyces cerevisiae using the amino terminal sequence of human IL-1β as a secretagogue. The combination of existing secretion readers and facilitating peptides increased the secretion of several proteins (Lee et al., Biotechnol. Prog. , 1999, 15: 884-890).

In a preferred embodiment of the present invention, the expression vector is a killer toxin leader sequence, the gene of the amino terminal 24 residues of interleukin 1β, the gene corresponding to the KEX2 protease cleavage site and the expression vector comprising the gene described in SEQ ID NO: 10 ( pIL20XEK _L -Ca-His). In addition, a transformant transformed with the expression vector (pIL20XEK _L- Ca-His) into yeast was prepared and deposited on May 21, 2002, at the Korea Microorganism Conservation Center (Accession No .: KCCM 10381). ).

In addition, in a preferred embodiment of the present invention, the expression vector comprises a killer toxin leader sequence, a gene of the amino terminal 24 residue of interleukin 1β, a gene corresponding to the KEX2 protease cleavage site, and an expression vector comprising the gene of SEQ ID NO: 18 . (pIL20-I1V-EK _L ). In addition, a transformant transformed with the expression vector (pIL20-I1V-EK _L ) into yeast was prepared, and deposited on May 21, 2002, at the Korea Microorganism Conservation Center (accession number: KCCM 10382). ).

The present invention also provides a method for producing a recombinant protein, wherein the recombinant enterokinase protein is separated using an affinity tag. In the present invention, a transformant expressing the recombinant enterokinase of the present invention is cultured, and the recombinant enterokinase is separated from the culture solution using an affinity tag. Affinity tags are separated using binding tags bound to recombinant enterokinase, and affinity tags include histidine tags, hemagglutinin (HA) tags, poly arginine tags, poly lysine tags, You can use flag tags, myc tags, and so on. In a preferred embodiment of the present invention, recombinant enterokinase was separated from the mass cultured yeast broth using an expanded bed adsorption (EBA) method to produce a recombinant enterokinase.

In addition, the present invention provides a method for producing a target protein that can increase the yield of the target protein by cutting the fusion protein using the recombinant enterokinase protein and recovering the recombinant enterokinase protein in the reaction solution to prevent degradation of the target protein to provide.

By using the recombinant enterokinase of the present invention in the cleavage of the fusion protein to increase the yield of the target protein

1) cleaving by adding the recombinant enterokinase of the present invention to the fusion protein;

2) after the cleavage reaction is completed, removing the recombinant enterokinase from the cleavage reaction solution.

In step 2, the removal of the recombinant enterokinase can be easily separated using a chromatography immobilized ligand binding to the affinity tag of the recombinant enterokinase.

Affinity tags have the property of having affinity with specific ligands. Recombinant enterokinase can be isolated easily by liquid chromatography using a resin with an affinity ligand. In addition, when the recombinant enterokinase is used for cleavage of the recombinant fusion protein, after the cleavage of the mixed solution of the fusion partner, the target protein, and the enterokinase after the cleavage, the enterokinase is adsorbed to the ligand, so the fusion fluid is fused to the flow. Only the partner and the target protein remain. In particular, in the case of a fusion partner having an affinity tag such as that of enterokinase, only the target protein can be obtained by passing through chromatography once. In addition, in the production of enterokinase using Escherichia coli, enterokinase expressed in the form of inactive aggregates can be obtained by high concentration of the active protein by immobilization and refolding on a chromatography column using an affinity tag.

The present inventors observe that if left unremoved in the cleavage reaction of the fusion protein without removing the recombinant EK _L , a smaller protein than the target protein is produced and the yield of the target protein is decreased, and the cause is contaminated when preparing enterokinase. As a result of confirming whether it is due to the proteolytic enzyme or the broad substrate specificity of enterokinase, it was confirmed that the further cleavage of the fusion protein is not by the contaminated protease but by the enterokinase itself. It was also found that recombinant EK _L and natural enterokinase (hereinafter abbreviated as "bEK") differ in substrate specificity. Because of its small size, rEK _L, which represents the catalytic subunit of the homoenzyme, is more likely to access difficult locations. In the prior art, further degradation of the target protein was observed when enterokinase was used for cleavage of the fusion protein, which has been considered to be due to contaminated protease in the separation of enterokinase (Dykes et al., Eur.J. Biochem ., 1988, 174: 411-416). However, the present invention demonstrates that this further degradation is not due to contaminated proteases but due to the broad substrate specificity of enterokinase itself. From the above results, it was found that it is essential to remove enterokinase after the cleavage reaction to prevent further degradation of the target protein.

The recombinant enterokinase protein of the present invention can be cultured from a transformant and purified in a single step by nickel affinity chromatography, batch adsoption, etc. The purified recombinant enterokinase exhibits complete activity.

This single step purification procedure is also used to remove enterokinase after cleaving the fusion protein to prevent unwanted cleavage of the protein of interest. In a preferred embodiment of the present invention rEK _L -Ca-His was easily removed by simple centrifugation with a nickel-NTA spin column, but recombinant EK _L without an affinity tag was not easily removed (see Figure 9 ). rEK _L -Ca-His can also be easily removed by batch adsorption. Nickel affinity chromatography has many advantages over benzamidine or soybean trypsin inhibition chromatography to remove His-tagged EK _L. Resins are stable, economical and very specific (Porath et al., Nature , 1975, 258: 598-599). In addition, histidine tags bind tightly to the metal-chelating matrix under a wide range of conditions such as high salts, surfactants and denaturants. Thus, cleavage reactions can be used in a wide range of conditions to suit the desired protein. In addition, enzymes can be removed directly from the reaction solution without the need for subprocesses such as dialysis or gel permeation. Histidine tags are the most well known affinity tags and are widely used for fusion proteins in prokaryotic or eukaryotic cells (Porath et al., Protein Expr. Purif ., 1992, 3: 263-281). His-tag EK _L is more beneficial for cleavage of His-tag fusion protein because it can obtain the desired protein from His-tag fusion protein and can remove EK _L in a single step in a nickel affinity column.

Accordingly, the carboxyl terminal His-tag EK _L of the present invention can be used in downstream processes for producing and purifying proteins from cultures and for recovery and recycling of enzymes during the reaction to be useful for maximizing product yield. Can be. In addition, the immobilization of rEK _L- Ca-His to the nickel affinity column is not a covalent bond but a reversible action. In addition, the rEK _L- Ca-His of the present invention enables the enzyme to exhibit full activity by minimizing the steric hindrance of the enzyme active site towards the column mattress with the specific orientation of the affinity tag. Therefore, the carboxyl terminal affinity tag enterokinase of the present invention can be very useful in fusion protein technology and can reduce the production cost of various recombinant proteins.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Reference Example 1 Recombinant EK _L Expression and Characterization of

<1-1> EK _L Preparation of expression vector comprising

The inventors have cloned by performing, PCR the EK _L cDNA in order to prepare an expression vector containing the EK _L

Specifically, all RNA was isolated from bovine duodenal tissue using an oligotex RNA kit (Qiagen) using the manufacturer's instructions. And from all RNA using the primers described in SEQ ID NO: 3 and SEQ ID NO: 4 corresponding to the amino terminus and carboxyl terminus of bovine EK _L perform RT-PCR to obtain cDNA encoding a bovine EK _L. PCR products were cleaved using restriction enzymes KpnI and HindIII and cloned into the ProFuse vector pGE-lysN (Proteon, Korea), and the base sequence of bovine EK _L was determined by Sanger et al., Proc. Natl. Acad, Sci. USA, 1977, 74: 5463-67).

As a result of analyzing the base sequence of EK _L , it was confirmed that enterokinase had two types of cDNA, one of which encodes the 235 amino acid residue represented by SEQ ID NO: 11 and the other of 107-138 32 amino acids of the portion were coding for the missing 203 amino acid residue. The cDNA sequence of EK _L was very similar to the clone reported in the previous literature (LaVallie et al., J. Biol. Chem ., 1993, 268: 23311-17), but the 615 position changed from A to G and the 669 position. There was a difference between the two parts where G changed to C.

To give the 32-residues deletion ΔEK _L corresponding to amino acids 107-138 parts of two forms of the enterokinase gene by PCR EK _L and _L EK as described above. The enterokinase gene was subjected to a second PCR using the primers described in SEQ ID NO: 5 and SEQ ID NO: 6 , and the PCR product was cleaved with restriction enzymes XbaI and BamHI to express the SecHancer vector pIL20GH (proteon, Korea). Was inserted into a pIL20EK _L plasmid and a pIL20ΔEK _L plasmid. In the plasmid, the enterokinase gene is linked to the carboxy terminal 24 residue of human interleukin-1β as a secretion promoter following the killer toxin leader sequence (Lee et al., Biotechnol. Prog. , 1999, 15: 884-890). The KEX2 protease cleavage site is located between the secretion promoter and EK _L ( FIG. 1 ). Gene expression is regulated by the Gal-10 active sequence on top of the S. cerevisiae α-Mating Factor promoter and induced by 2% galactose.

<1-2> EK _L Preparation of a transformant transformed with an expression vector comprising a

The present inventors prepared a transformant transformed with an expression vector comprising EK _L prepared in Reference Example <1-1>. Specifically, the expression vector pIL20EK _L plasmid and pIL20ΔEK _L plasmid prepared in Reference Example <1-1> were transferred to Saccharomyces cerevisiae 2805 (pep4 :: HIS3, pro1-δ, can1, Gal2). , his3δ, ura3-52) were transformed. The yeast transformants were prepared in the same manner as in the previous technique (Lee et al., Biotechnol. Prog ., 1999, 15: 884-890) in SD Ura medium (0.8 g / L complete supplement medium (BIO101), without amino acids. 6.7 g / L yeast nitrate (DIFCO), 2% glucose) was incubated overnight at 30 ° C., 250 rpm. The culture was centrifuged to induce expression by suspending the cell precipitate in YPGal medium (1% yeast extract, 2% bacto-peptone, 2% galactose). Expression induction continued at 30 ° C. at 250 rpm for 20 hours.

<1-3> EK _L Expression and Purification

The present inventors cultured the transformant prepared in Reference Example <1-2> to express recombinant EK _L , and purified the recombinant EK _L protein at once with a benzamidine sepharose column. Specifically, the yeast culture containing the secreted enterokinase was dialyzed in buffer A (50 mM Tris-HCl, pH8.4) and pre-filled with buffer A (benzamidine Sepharose column, Phamacia, 1.6). Cm x 5 cm). At this time, the flow rate was adjusted to 20 ml / h. The column was washed with 100 mL buffer A and then again with 50 mL 100 mM Tris-HCl, pH 8.4. Recombinant EK _L was eluted with 50 vol% ethylene glycol in 0.5 M Tris / Acetate buffer at pH 5.6 with 1 M NaCl. Fractions with enterokinase activity were selected and concentrated in dialysis tubes with gum Arabic, Sigma, and the last fraction was dialyzed overnight with 50 mM Tris-HCl 4 liters at pH 8.0.

As a result, about 1 mg of EK _L was obtained from 1 liter of culture medium. As a result of analyzing the sequence of the purified protein, it was confirmed that the carboxyl terminal was correctly cleaved by KEX2 protease during secretion. The expected molecular weight of EK _L was 26.3 kDa, but the molecular weight of EK _L measured on SDS-PAGE was 44 kDa because it was glycosylated. Treatment of the recombinant EK _L with endoglycosidase H cutting mannose glycans yielded EK _L at a position lower than 44 kDa ( FIG. 2 ).

<1-4> EK _L Fusion protein cleavage using

We digested the fusion protein (T2GH) (Shin et al, 1998, J. Biotechnol. 62: 143-151) with recombinant EK _L and purified on a benzamidine sepharose column.

As a result, a human growth hormone (hGH) product having 22 kDa could be identified on SDS-PAGE. Sequencing the amino terminus of the cleavage product showed that the amino acid sequence (Asp) ₄ -Lys recognition sequence described in SEQ ID NO: 2 of the fusion protein was correctly cleaved. However, by continuing to react the reaction mixture, a protein of lower molecular weight than the original was observed with the decrease of the hGH protein ( FIG. 3A ).

<1-5> recombinant EK _L Substrate specificity

In the reference example <1-4>, the present inventors observed that if the recombinant EK _L was continuously removed after cleavage of the fusion protein, a protein smaller than the target protein was produced and the yield of the target protein was decreased. In order to determine whether the cause is caused by contaminating proteolytic enzymes or by the broad substrate specificity of enterokinase when separating enterokinase, the fusion protein may be used in various forms of yeast transformant fermentation supernatant or Treatment with natural enterokinase derived from bovine duodenum (hereinafter abbreviated as "bEK").

First, since there is a possibility that other protease is contaminated in the process of preparing a recombinant EK _L purified using a benjamidine sepharose column, the present inventors used a fermentation supernatant cultured enterokinase. Three different fermentation supernatants, y (pIL20X), y (pIL20GH) or y (pIL20ΔEK _L ), used as negative controls, each produced no protein or internally deficient EK _L without hGH or no activity. .

As a result, cleavage of the fusion protein by bEK resulted in a product of 22 kD molecular weight corresponding to human hGH and fragment A of smaller size ( FIG. 3B , lane 3 and lane 4). The same treatment as above with the fermented supernatant containing recombinant EK _L produced fragment A and fragment B ( FIG. 3b , lane 8). No cleaved product was observed in the three negative controls ( FIG. 3B , lanes 5-7). The results indicate that the further cleavage of the fusion protein is not by contaminated protease but by enterokinase itself. In addition, fragment B was not detected by treatment with bEK but only by cleavage with recombinant EK _L. From the above results, it was found that recombinant EK _L and bEK had different substrate specificities.

Because of the small size of rEK _L , which represents the catalytic subunit of the homoenzyme, it is more likely to access difficult locations. The results of the present invention indicate that the activity of bEK is higher than that of rEK _L in activating trypsinogen. Consistent with the previous technique (LaVallie et al., J. Biol. Chem ., 1993, 268: 23311-17), which is about 100 times higher and rEK _L has a higher activity of cleaving fusion proteins than bEK. In the previous technique, further degradation of the target protein was observed when enterokinase was used for cleavage of the fusion protein, which has been considered to be due to contaminated protease in the separation of enterokinase (Dykes et al., Eur.J. Biochem ., 1988, 174: 411-416). However, the present invention demonstrates that this further degradation is not due to contaminated proteases but due to the broad substrate specificity of enterokinase itself.

From the above results, it was found that enterokinase must be removed after the cleavage reaction to prevent further degradation of the target protein.

Example 1 His-Tag EK _L Preparation of expression vector comprising

In order to facilitate the purification and removal of recombinant EK _L after cleavage of the fusion protein, the present inventors inserted a histidine tag into the amino or carboxyl terminus of the recombinant EK _L , and thus the amino-terminal His-tag EK _L (hereinafter referred to as "His-N- rEK _L "is called or carboxyl terminal His-tag EK _L (hereinafter" rEK _L -Ca-His ") was prepared.

Specifically, PCR was performed on the pIL20EK _L vector prepared in Reference Example <1-1> using the primers described in SEQ ID NO: 6 and SEQ ID NO: 7 , and then the amino-terminal His-tagged EK _L gene was converted into the pIL20GH vector. The pIL20X-N-His-EK _L plasmid was prepared by cloning at the XbaI / BamHI position. In addition, PCR was performed using primers described in SEQ ID NO: 5 and SEQ ID NO: 8 , and then the carboxyl His-tag EK _L gene was cloned into the pIL20GH vector to prepare pIL20XEK _L -Ca-His plasmid. N-His-EK _L and EK _L -Ca-His are expressed as His ₆ -D ₄ K-EK _L and EK _L -His ₅ , respectively. The number of histidine tags may vary depending on the application and is not limited to the experiment.

<Example 2> Preparation of transformants transformed with the expression vector

The present inventors prepared a transformant in the same manner as in Reference Example <1-2> using the expression vector prepared in Example 1. Specifically, the expression vectors N-His-EK _L and rEK _L- Ca-His plasmids prepared in Example 1 were prepared in S. cerevisiae 2805 (pep4 :: HIS3, pro1-δ, can1, Gal2, his3δ, ura3). -52). Yeast cells were prepared in the same manner as in the previous technique (Lee et al., Biotechnol. Prog ., 1999, 15: 884-890) in SD Ura medium (0.8 g / L complete supplement medium (BIO101), 6.7 g / without amino acids. Incubated overnight in 10 ml L yeast nitrate (DIFCO) and 2% glucose) at 30 ° C. at 250 rpm. The culture was centrifuged to induce expression by suspending the cell precipitate in YPGal medium (1% yeast extract, 2% bacto-peptone and 2% galactose). At this time, expression induction was continued for 20 hours at 30 ° C. and 250 rpm.

The present inventors deposited a transformant containing the carboxyl terminal His-tag EK _L gene with the Korea Microorganism Conservation Center, an international depository institution, on May 21, 2002 (Accession No .: KCCM 10381).

Example 3 rEK _L -Ca-His Purification

The present inventors have found that by using a His- tag located in the carboxyl terminal of rEK -Ca _L-His was purified rEK -Ca _L-His of the present invention in a single step from Nickel chelating Sepharose chromatography. Specifically, the expression solution was dialyzed in 20 mM sodium phosphate (pH 7.5), 300 mM NaCl (abbreviated as buffer B), which was flowed into a nickel chelating Sepharose column (1.6 cm X 5 cm), and then to buffer B. Washing was performed again with a solution of 50 mM imidazole added to the above buffer B, and the protein was eluted with buffer B added with 100 mM imidazole. The eluted solution was collected by only investigating protein activity and concentrated with Centriprep 10 (Amicon) to isolate proteins.

As a result, the tablet yield was 1 mg per liter of fermentation supernatant. Enterokinase activity was observed in fractions extracted from buffer containing 100 mM imidazole. First washing with a buffer containing 50 mM imidazole did not extract most of the enterokinase but extracted a host-derived protein of about 47-50 kDa size ( FIG. 4 , lane 2). Since His-N-rEK _L was not purified as efficiently as EK _L- Ca-His under the same conditions, the present inventors used the partially purified product for cleavage reaction. As a result, it was found that recombinant enterokinase whose carboxyl terminus of the enterokinase light chain is histidine-affinity tagged is industrially useful. However, this study revealed that the carboxyl terminal is exposed to the outside without affecting the activity of the enzyme, so it is possible to use all of the affinity tags identified so far, not limited to histidine tags.

Example 4 rEK _L Characterization of -Ca-His

We used rEK _L -Ca-His to cleave the fusion protein and also to determine whether carboxyl or carboxyl modification of EK _L affects enzymatic activity. _The activities of _L, His-N-rEK _L and rEK _L- Ca-His plasmids were compared.

Enzyme activity was determined by fluorescence assay (Grant and Herman-Taylor, Biochim. Biophys. Acta ., 1979, 16: 207-215), and protein concentration in recombinant EK _L solution was determined by micro bosa serum albumin (BSA) assay. It was. Specifically, the enzyme was added to a 50 uM concentration of the fluorescent substrate Gly- (Asp) ₄ -Lys-β-naphthylamide solution dissolved in a mixture of 70 mM Tris-HCl and 10% DMSO (pH 8.0). Fluorescence was measured at excitation at 337 nM and emission at 420 nM, and enzyme activity was calculated from changes in fluorescence over time.

As a result, the activity of rEK _L -Ca-His increased with time similar to recombinant EK _L , but His-N-rEK _L could not detect a detectable activity ( Fig. 5 ). From the above results, it was found that the properties of amino-terminal residues rather than carboxyl-terminals play an important role in enterokinase activity. The amino-terminal amino acid isoleucine of recombinant EK _L is involved in salt bridges that are crucial in the protein structure and thus extending the amino-terminal inhibited the activity of the protein (Fehlhammer et al., J. Mol. Biol ., 1977, 11: 415-438; Collins-Racie et al., Bio / technology , 1995, 13: 982-987). In fact, the three-dimensional structure of recombinant EK _L is that the carboxyl terminal isoleucine residues are located inside the protein for the salt bridge, while the carboxyl terminal sites are external (Lu et al., J. Mol., Biol ., 1999, 292: 361-373). From the above results, it was confirmed that the structure known to the expected role of the amino terminal.

Example 5 Preparation and Selection of Enterokinase Mutants with Modified Amino Terminals

His-N-rEK _L was devoid of EK _L activity (see FIG. 5 ) . To investigate the effect of the amino terminus of the enterokinase light chain on the enzyme, an amino terminal-modified enterokinase mutant was first prepared and selected. Mutant enterokinase expression plasmids with modifications at the amino terminus of enterokinase were prepared. pIL20EK _L -Ca-His as a template for the substitution of amino-terminal amino acids with other amino acids, addition of methionine, insertion of 5 amino acids with (Asp) _4- Lys sites, and removal of amino-terminal amino acids It was prepared using enzyme chain reaction. Primer sequences used for the preparation of each mutation are as follows. Primer 1, set forth in SEQ ID NO: 12 , was an antisense primer and was used identically for all mutation preparations. Primer 2, set forth in SEQ ID NO: 13 for amino terminal amino acid (isoleucine) substitution, prepared a primer using NNN as the codon of amino terminal amino acid. Polymerase chain reaction (PCR) was carried out using primers described in SEQ ID NO: 12 and SEQ ID NO: 13 to obtain mutated DNA substituted with 17 different amino acids through sequencing. Mutants added with methionine at the amino terminus are primers set forth in SEQ ID NO: 12 and SEQ ID NO: 14 ; mutations incorporating five amino acids and the (Asp) ₄ -Lys site; primers set forth in SEQ ID NO: 12 and SEQ ID NO: 15 , and amino Mutations from which the terminal amino acids were removed were obtained by polymerase chain reaction using primers described in SEQ ID NO: 12 and SEQ ID NO: 16 .

The DNA obtained by performing the PCR was treated with restriction enzymes BamHI and XbaI and inserted into the pIL20 vector using T4 ligase, thereby expressing each mutant expression vector (pIL20Xi-EK _L (i = 1-17), pIL20Metα-EK _L , pIL20Ex-EK _L , pIL20ΔIle-EK _L ) were prepared ( FIG. 6 ).

Yeast strain (Saccharomyces cerevisiae 2805) was transformed with each expression vector prepared above. Transformed yeast strains were inoculated in 10 ml SD Ura-medium (0.8 g / L Complete Supplement Media (BIO101), yeast nitrogen salt without 6.7 g / L amino acid (DIFCO), 2% glucose) at 30 ° C. for 24 hours. Shake culture at 200 rpm speed. The pellet was centrifuged and the pellet was again suspended in induction medium (1% yeast extract, 2% Bacto-peptone, 2% galactose), and then cultured at 30 rpm for 24 hours at 200 rpm. The supernatant was collected by centrifugation of the culture solution, and the enzyme activity was measured using it, and the expression level was examined by Western blotting. Enzyme activity was measured using fluorescent spectrophotometer using fluorescent substrate Val- (Asp) ₄ -Lys-β naphthylamide (Sigma) to generate β-naphthylamine that fluoresces with time. It was. 0.5 mM substrate was mixed in 25 mM Tris-HCl (pH 8.4), 10 mM CaCl ₂ , 10% DMSO solution, and the culture supernatant was kept at 37 ° C. while maintaining λex = 337 nm and λem = 420 nm wavelength. It was measured that the fluorescence increased for 5 minutes with the light of.

As a result, 17 mutants substituted with amino terminal amino acids, mutants added with methionine, 5 amino acids and (Asp) -inserted _4- Lys sites, mutants without amino terminal amino acids, and a total of 20 mutations were almost completely expressed. Enzyme activity was seen only in mutations in which enterokinase and amino terminal amino acids were substituted with valine without genetic modification ( Table 1 ).

The present inventors named the mutant expression vector "pIL20-I1VEK _L " in which the enterokinase amino terminus was replaced with valine in isoleucine, and transformed the expression vector pIL20-I1V-EK _L into yeast. It was manufactured and deposited on May 21, 2002, to the Korea Microorganism Conservation Center, an international depository institution (Accession No .: KCCM 10382).

Construct Expression level activation Construct Expression level activation pIL20X1EK _L (I1A) + - pIL20X11EK _L (I1M) ++++ - pIL20X2EK _L (I1R) + - pIL20X12EK _L (I1F) ++++ - pIL20X3EK _L (I1C) +++ - pIL20X13EK _L (I1P) +++ - pIL20X4EK _L (I1Q) +++ - pIL20X14EK _L (I1S) + - pIL20X5EK _L (I1E) +++ - pIL20X15EK _L (I1T) ++++ - pIL20X6EK _L (I1G) + - pIL20X16EK _L (I1Y) ++ - pIL20X7EK _L (I1H) ++ - pIL20X17EK _L (I1V) ++++ +++++ pIL20X8EK _L (WT) ++++ +++++ pIL20Metα-EK _L ++ - pIL20X9EK _L (I1L) ++++ - pIL20Ex-EK _L ++ - pIL20X10EK _L (I1K) +++ - pIL20ΔIle-EK _L ++++ -

Example 6 Comparison of Characteristics of I1V Mutation and Wild Type Enterokinase

The present inventors analyzed the enzymatic activity of I1V mutant enterokinase and wild type enterokinase wherein the enterokinase amino terminus was replaced with valine in isoleucine.

As a result, I1V mutants and wild type enterokinase obtained under the same conditions showed almost similar activity when compared to enzyme activity ( FIG. 7 ). In order to determine the stability according to the temperature of the protein, the relative enzyme activity was measured with time at 45 ° C. and 65 ° C. and showed a similar pattern ( FIG. 8 ).

Since six histidines added to the carboxyl terminal adsorb the nickel, each enzyme was purified from the culture using a Ni-NTA spin column (Qiagen). The culture was concentrated and dialyzed in a solution of 300 mM NaCl, 10 mM imidazole in 50 mM sodium phosphate (pH 8.0) buffer and passed through a Ni-NTA spin column. The adsorbed protein was eluted with a solution of 250 mM imidazole added to the above solution. The two purified proteins were compared with each other by comparing the activity of the substrate with the concentration of K _m and k _cat for each enzyme ( Table 2 ).

enzyme Origin K _M (mM) k _cat (s ^-1 ) k _cat / K _M (M ^-1 s ^-1 ) references Purified Enteropeptidase (Dimer) Human 0.28 28.3 101000 (One) Purified Enteropeptidase (Dimer) Bovine 0.22 24.0 109000 (2) Purified Enteropeptidase (Dimer) Bovine 0.20 16.7 83000 (3) Recombinant Protein Expressed in L-BEK Escherichia Coli Bovine 0.61 42.7 70400 (4) Recombinant Protein Expressed in rEK _L Yeast Bovine 0.78 28.0 35897 The present invention Recombinant Protein Expressed in I1V Mutant rEK _L Yeast Bovine 0.69 40 57971 The present invention

References (1) Grant, DAW, and Hermon-Taylor, K. (1979) Biochim. Biophys. Acta 567; 207-215; (2) Lu, D. et al., (1997) J. Biol. Chem . 272; 31293-31300; (3) Mikhailova, AG and Rumsh, LD (1999) FEBS Letters 442; 226-230; (4) Lu, D. et al., (1999) J. Mol. Biol . 292; 361-373

As a result, the I1V mutant enterokinase was almost similar in enzyme activity compared to wild type enterokinase. In order to produce enterokinase on a large scale, E. coli production should be possible. However, when expressed using E. coli, the amino terminal isoleucine, which is very important for enzyme activity, is not well exposed by methionine amino peptidase and methionine is added. Enzyme activity is lost. However, since methionine amino peptidase has a property of easily cleaving when the amino terminal is substituted with valine, I1V mutant enterokinase has an advantage that it can be applied to mass production of enzymes using E. coli.

Example 7 rEK _L Removal of Ca-His

The present inventors used rEK _L -Ca-His to cut the fusion protein, and then removed rEK _L -Ca-His so that no further degradation of the target protein occurred. Specifically, 1 μg of the isolated protein was mixed with 500 μl of buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 50 mM imidazole, pH 8.0), loaded onto a nickel NTA-spin column, and then centrifuged at 700 g for 2 minutes to eluate. The enzyme activity of was compared.

As a result, carboxyl terminal His-tagged rEK _L was removed by nickel affinity chromatography in a single step in the culture medium, but showed little activity, but the recombinant EK _L was not removed and showed high activity ( FIG. 9 ).

From the above results, the carboxyl terminal His-tag EK _L of the present invention is useful for maximizing the yield of the product by being used in the downstream process of producing and purifying proteins from the culture and the recovery and recycling of enzymes during the reaction. Can be used. The fixation of rEK _L- Ca-His to the nickel affinity column is not a covalent bond but a reversible action. In addition, the rEK _L- Ca-His of the present invention enables the enzyme to exhibit full activity by minimizing the steric hindrance of the enzyme active site towards the column mattress with the specific orientation of the affinity tag.

Therefore, the carboxyl terminal affinity tag enterokinase of the present invention can be very useful in fusion protein technology and can reduce the production cost of various recombinant proteins.

Example 8 rEK Using EBA Method _L -Ca-His Purification

The inventors separated rEK _L -Ca-His and I1V-EK _L from the mass cultured yeast cultures quickly and simply using EBA (Expanded bed adsorption) method. Specifically, the pH was adjusted by adding 20 mM sodium phosphate (pH 8.0) to a culture medium in which a large amount of transformants expressing rEK _L -Ca-His or I1V-EK _L were cultured using a fermenter, and then nickel-filled. (charge) A chelating Sepharose EBA column (Streamline 25, Amersham-Pharmacia Biotech Inc.) was passed upwards from below (flow rate: 300 cm / hr). Washed in the same direction with 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, 10 mM imidazole solution and eluted by flowing 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, 300 mM imidazole solution in the opposite direction. . The protein in the culture was effectively obtained in about 87% yield.

This method is an effective method to prevent the protein denaturation due to heat or foam generation during the concentration process, saving the time and cost of precipitating and removing cells from the culture medium and concentrating the protein secreted into the culture medium. It is thought that the development and development of recombinant enterokinase by making it possible to concentrate and isolate the protein in a single step and to easily scale up (scale up).

As described above, the recombinant enterokinase protein modified by the carboxyl or amino terminus of the enterokinase light chain of the present invention can be easily recovered and removed after protein separation and cleavage of the fusion protein, and effective column immobilization will be possible. In addition, since it is possible to mass-produce in E. coli, it can be usefully used to produce the target protein from the fusion protein.

Claims (14)

Recombinant enterokinase protein wherein the amino or carboxyl terminus of the enterokinase light chain is modified. The recombinant enterokinase protein according to claim 1, wherein the recombinant enterokinase protein of which the carboxyl end is modified is added to a carboxyl end of the light chain of the enterokinase. The method of claim 2, wherein the affinity tag is a histidine (His) tag, a hemagglutinin (HA) tag, a poly arginine tag, a poly lysine tag, a flag tag, and a myc tag. Recombinant enterokinase protein, characterized in that selected from the group consisting of. 4. The recombinant enterokinase protein of claim 3, wherein said recombinant enterokinase protein comprises an amino acid sequence set forth in SEQ ID NO: 9 . The recombinant enterokinase protein according to any one of claims 1 to 4, wherein the recombinant enterokinase protein wherein the amino terminus is modified is substituted with valine of isoleucine at the amino terminus of the light chain of enterokinase. The recombinant enterokinase protein of claim 5, wherein the recombinant enterokinase protein comprises an amino acid sequence set forth in SEQ ID NO: 17. 7 . A gene comprising the nucleotide sequence set forth in SEQ ID NO: 10 encoding the recombinant enterokinase protein of claim 4. An expression vector comprising a killer toxin leader sequence, a gene of an amino terminal 24 residue of interleukin 1β, a gene corresponding to a KEX2 protease cleavage site, and a gene described in SEQ ID NO: 10 . A transformant transformed with the expression vector of claim 8 (accession number: KCCM 10381). A gene having the nucleotide sequence set forth in SEQ ID NO: 18 encoding the recombinant enterokinase protein of claim 6. An expression vector comprising a killer toxin leader sequence, a gene of 24 amino terminal residues of interleukin 1β, a gene corresponding to a KEX2 protease cleavage site, and a gene described by SEQ ID NO: 18 . A transformant transformed with the expression vector of claim 11 (accession number: KCCM 10382). In the method for preparing a target protein by cleaving the fusion protein, the recombinant enterokinase protein of claim 1 is added to the fusion protein and reacted. Method for producing a protein of interest, characterized in that. A method of culturing a transformant expressing the recombinant enterokinase of claim 1, and separating the recombinant enterokinase from the culture using an affinity tag.