KR0166424B1 - Preparation method of target protein from fusion protein using urokinase - Google Patents

Abstract

The present invention expresses a specific amino acid sequence cleaved by urokinase between the target protein of the fusion protein and the fusion partner, and cleaves the expressed fusion protein with urokinase, thereby cutting the target protein from the fusion protein. It relates to a manufacturing method. In the present invention, when the amino acid sequence of the urokinase cleavage site located between the target protein and the fusion partner of the fusion protein is -X-Gly-Arg (X is Pro, Thr, Ile, Phe or Leu), the cleavage efficiency is excellent. It was also found that the expression of the fusion protein including the cleavage site was excellent, and it was confirmed that by using the amino acid sequence of the urokinase cleavage site determined in the present invention, it was possible to economically and easily manufacture a large amount of the protein from the fusion protein.

Description

Method for preparing target protein from fusion protein using urokinase

Figure 1 shows the sequencing of the mixed oligonucleotides used for site specific mutations in the present invention and the imino acids translated therefrom.

2 is a schematic diagram illustrating the process of constructing plasmid ΔpLEK-2-UP.

3 shows a genetic map of the expression vector ΔpMA.

Figure 4 is a photograph showing the results of the SDS-PAGE of each sample in the process of separating the GSTt / UK / PTH fusion protein from the E. coli culture transformed with the expression vector containing the GSTt / UK / PTH gene.

5 is a photograph showing the results of SDS-PAGE after reacting various mutants of GSTt / UK / PTH fusion proteins with or without adding urokinase.

FIG. 6 measures the cleavage efficiency of the Gly-Thr-Gly-Arg site by urokinase and compares each reactant with electricity to compare the cleavage efficiency of the Ile-Glu-Gly-Arg site by Factor Xa. The photograph shows the result of mobilization.

The present invention relates to a method for preparing a protein of interest from fusion proteins using urokinase. More specifically, the present invention expresses a specific amino acid sequence cleaved by a serine protease, urokinase, between the target protein of the fusion protein and the fusion partner, and the expressed fusion protein. The present invention relates to a method for producing a protein of interest from a fusion protein by cleaving with a urokinase.

With the development of recombinant DNA technology, a process for mass expression of various useful proteins using a microbial expression system and industrialization thereof has been developed. In particular, in order to increase the expression efficiency of foreign proteins, a method of expressing a target protein using a protein having excellent expression in a host microorganism to be used as a fusion partner is mainly used, wherein the target protein is separated from the expressed fusion protein. Is one of the most important factors affecting the production cost and the purity and yield of the final purified protein (Fiona AO Marston, Biochem, J., 240: 1-12 ( 1986)).

In general, a method of cleaving a target protein from a fusion protein can be broadly classified into a chemical cleavage method using an chemical substance and an enzymatic cleavage method using an enzyme. Among these, the cutting method using chemicals has the advantage of cost savings because it uses inexpensive chemicals, but because the specificity of the cutting site is low, various by-products other than the target protein are produced during cutting, so these by-products Since the process for separating and purifying the target protein has a further disadvantage. On the other hand, the cleavage method using enzymes can solve the problem of the cleavage method using chemicals because of the specificity of the cleavage site, but the cost of the enzyme used is expensive, so it is a problem to use this method on an industrial scale. There is.

As a result, most researchers studying the mass production process of the synthesized protein have attempted to economically use enzymes to cleave the protein of interest from the fusion protein. However, the proteolytic enzymes Factor Xa, thrombin, and enterokinase, which are currently developed and used by the neck, have a limitation in obtaining a large amount, and thus, a cleavage method using an enzyme Despite its many advantages, it is not widely used at the industrial level.

Therefore, there is an urgent need for a third enzyme that can be economically mass-produced which can be efficiently used in an enzyme cleavage method.

Therefore, the inventors of the present invention have already developed a mass production process for the two-chain urokinase type plasminogen activator, a serine-based protease that is used as a thrombolytic agent, and especially of prokaryotes such as E. coli. It is known that the expression system can be used to mass-produce active urokinases (WEHolmes et al., Bio / Techonology, 3: 923-929 (1985)), and other enzymes such as electrofactor teneigen. In view of the fact that a large amount of urokinase can be produced and obtained more economically, it has been devoted to a method for preparing a target protein from a fusion protein using urokinase.

On the other hand, urokinase is a serine protease similar to factor tenei and thrombin, and is known to cleave carboxyl-side imide bonds of basic amino acid residues such as arginine and lysine. Studies on substrate specificity of urokinase have been conducted mainly by using chemically synthesized peptides as substrates. To date, the peptide of pyroGlu-Gly-Arg is known as the most efficient recognition and cleavage site of urokinase.

In view of this, in the following structural formulas of pyroGlu and proline, since pyroGlu has the most similar structure to the proline amino acid except the carbon position 5, Pro-Gly-Arg is the protein of urokinase. It can be expected to be the most efficient recognition and cutting site.

However, comparing the carbonyl (-CO-) group at the 5th carbon position of pyroGlu with the -CH ₂ -group of the proline at the same position, the hydrophillcity as well as the space occupied by each group Because of the different properties in various aspects, such as hydrogen bonds (hydrogen bonds), it is not possible to rule out the possibility that the proline has a significantly different property in the protein than the chemically synthesized pyroGlu.

Therefore, the inventors of the present invention sought to determine the amino acid sequence that is the specific cleavage site by urokinase in the actual protein, -X, which is the amino acid sequence of the urokinase cleavage site specific between the target protein and the fusion partner of the fusion protein -Gly-Arg (X is Pro, Thr, Ile, Phe or Leu) in the presence of excellent cleavage efficiency, it was found that also excellent expression efficiency of the fusion protein including the cleavage site, and completed the present invention.

Finally, the main object of the present invention is to identify amino acid sequences in proteins that are recognized and cleaved by urokinase and to cut the amino acid sequence with urokinase so that the amino acid sequence is expressed between the fusion protein and the fusion partner, thereby removing the protein from the fusion protein. To provide an economical and high yield method.

It is another object of the present invention to provide a protein of interest prepared by the method of electricity.

Hereinafter, the present invention will be described in more detail.

The present inventors first focused on the fact that urokinase cleavage sites are similar to factor teneigen, and attempted to determine the cleavage sites of urokinase in proteins by manipulating DNA corresponding to cleavage sites of factor teneige: first, glutathione S transferase. Fragment of glutathione S transferase (hereinafter referred to as GSTt) gene and parathyroid hormone (PTH) gene which contains only 133 amino acids from the amino terminus among 235 amino acids of glutathione S transferase A template of a fusion protein having a cleavage site (Ile-Glu-Gly-Arg) gene (hereinafter referred to as GSTt / FX / PTH) as a template was used as a template. Site-directed mutagenesis is performed to replace Glu corresponding to the P3 subsite with another amino acid in Ile-Glu-Gly-Arg. The variant was prepared.

On the other hand, the fusion protein expressed from the microorganism transformed with the mutant was isolated and analyzed for the effect of urokinase cleavage. As a result, the amino acid sequence of the cleavage site was -X-Gly-Arg (X is Pro, Thr, Ile, Phe or Leu), the cutting efficiency is excellent, and in particular, -Thr-Gly-Arg cutting site was confirmed that the most efficient cutting by urokinase.

In addition, as a result of comparing the cutting efficiency of factor tenei and urokinase, the hourly cutting efficiency of -Thr-Gly-Arg cutting site by eurokinase was compared to the hourly cutting efficiency of Ile-Glu-Gly-Arg cutting site by factor teneige. It was increased by at least 10 times. Therefore, by using the amino acid sequence of the urokinase cleavage site determined in the present invention, it was expected that a large amount of the target protein can be produced from the fusion protein economically and easily.

The method for producing the protein of interest from the fusion protein using the specific cleavage site of the urokinase of the present invention will be widely used for the production of useful proteins.

Hereinafter, the present invention will be described in more detail by examples. It will be apparent to those skilled in the art that these embodiments are only for explaining the present invention, and the scope of the present invention is not limited to these examples. In particular, the present invention is not limited to the case of the fusion protein of GSTt and PTH described in the examples, and may be sufficiently applied to the case of the fusion protein containing various target proteins according to the object of the present invention.

Example 1

Factor Ten-Age Deformation

Since urokinase is known to have a similar cleavage site with factor tenae, it was intended to determine the cleavage site of urokinase in proteins by modifying the cleavage site of factor tenae. That is, the cleavage site Ile-Glu- of the factor tenei was formed by using GSTt / FX / PTH, a gene of the fusion protein, wherein the cleavage site of the factor tenei (Ile-Glu-Gly-Arg) is located between GSTt and PTH. In order to replace Glu corresponding to P3 subsite in Gly-Arg with any of the following amino acids, site-specific mutation was performed.

Inferred from the synthetic peptite pyroGlu-Gly-Arg-pNA, which exhibits the highest reactivity with respect to eurokinase, and is substituted with pororin, a natural amino acid having the structure most similar to pyroGlu:

Substitution with threonine having a hydroxyl group expected to exhibit oxygen-like properties and functions of the carbonyl group in pyroGlu:

(3) Substitution with isoleucine, phenylalanine or leucine, which are supposed to have hydrophobicity similar to proline:

In addition, site-specific mutations were performed such that isoleucine corresponding to the P4 subsite was replaced with glycine or side chain alanine having a small side chain so that the cleavage site was easily exposed to urokinase. For the site-specific mutations, mixed oliginucleotides including base sequences corresponding to each of the amino acids to be substituted were used (see FIG. 1). In FIG. 1, the 5 ′ first base of the codon corresponding to Glu in the cleavage region of the factor tenei is A, C or T; The second base is C or T; By using oligonucleotides with a third base of T, Glu was substituted with Pro, Thr, Ile, Leu, Phe or Ser.

To enhance the DNA of single-stranded GSTt / FX / PTH required to carry out the site-specific mutation, the GSTt / FX / PTH gene was isolated from ΔpLEK-2-FP and plasmid pBluescript or (+) ( Stratagene, USA). E. coli CJ236 (E. coli CJ236, F ′ cat (= pCJ105; M13 ^a Cm ^r ) d ung1 spot1 mcrA) transformed with this plasmid was infected with helper phage R408 to prepare uracil-containing single-stranded DNA. Then, site-specific mutations were performed using the mixed oligonucleotides of FIG. 1 by the method of Kunkel et al. (Kunkel et al., Proc. Natl. Acad, Sci, USA, 82: 488-492). (1985)), and by the method of Sanger et al. (Sanger et al., Proc. Natl. Acad. Sci., USA, 74: 5463 (1977)) substituted with a desired base sequence. It was confirmed. The substituted genes were named 'GSTt / UK / PTH', and the plasmid containing the former GSTt / UK / PTH gene was named ΔpLEK-2-UP. 2 shows a process of constructing? PLEK-2-UP.

Example 2

Preparation of Expression Vector ΔpMA

Chromosomal DNA was extracted from Salmonella typhymurium LT2 strain, treated with EcoRI restriction enzyme, and inserted into pUC19 vector to prepare pUC-Salmonella library. pUC-Salmonella library was introduced into E. coli DH5α strain (F'endA1 hsdR17 (rk-mk ⁺ ) supE44 thi-1 recA1 gyrA (NaF) U169Δ (lacZAY-argF) deoR) to obtain transformed E. coli colonies. Meanwhile, 5'-GCCATCGTCTTACTC-3 ', which is 15mer, and 5'-, which is 15mer for the nucleotide sequence which may include the araB-C regulatory region and the amino acid (N-terminal) third amino company (Glu) of the araC protein GCGTTTCAGCCATG-3 'oligonucleotides were synthesized. These were used as probes and colony hybridization was performed to select clones containing the araB-A and araC genes from the previously prepared pUC-Salmonella library.

2.52 Kb DNA fragment obtained by treating AvaI / SalI restriction enzyme with the selected plasmid pUC19-ara; And a 3.18 kb DNA fragment obtained by treating HindIII / SalI restriction enzyme to pUC119 (Maniatis et al., Molecular Cloning 2nd ed., 1989) vector with T ₄ DNA ligase, a UC 5.7 pUC119-araBC vector. Was prepared. After obtaining single-stranded DNA, 5'-CATATG-3 ', a nucleotide sequence cut by NbeI restriction enzyme, is inserted into the translation initiation code of the araB protein structural gene located at the bottom of the araB promoter, and the shine- of the araB promoter is inserted. The Dalgano sequence was modified to 5'-TAAGGAGG-3 ', resulting in site specific mutations. The modified ara clone was further treated with EcoRI-PvuII restriction enzyme to obtain a 2.61 kb DNA fragment containing the araB-C gene. Meanwhile, NdeI restriction enzyme was treated to Mpkl10 vector containing 228bp polyclonal region derived from pUC18, blunt-terminated with Klenow enzyme, recombined with T ₄ DNA ligase, and then treated with EcoRI / PvuII restriction enzyme. A DNA fragment of 2.7 kb was obtained. A 2.61 kb DNA fragment and a 2.7 kb DNA fragment were linked to each other, and an NcoI restriction enzyme site was inserted in the middle of the NbeI-EcoRI restriction enzyme site to prepare an expression vector ΔpMA of about 5,32 kb. : Republic of Korea Patent Application No. 94-11449). The gene map of ΔpMA thus prepared is shown in FIG.

Example 3

Expression of GSTt / UK / PTH Fusion Proteins and Isolation of Inclusion Body

The genes of GSTt / UK / PTH were separated from the ΔpLEK-2-UP prepared in Example 1 and subcloned into the expression vector ΔpMA prepared in Example 2, followed by E. coli MC1061 (E. coli MC1061, F-araD139 Δ (ara-leu) 7696 galE15 galK16 Δ (lac) X74rpsL (Str ^r ) hsdR2 (rk-mk ⁺ ) mcrA mcrB1) was transformed. Each transformant thus prepared was incubated overnight in LB / Amp medium (containing 10 g bacto-trypton, 5 g yeast extract, 10 g NaCl and 50 μg / ml ampicillin per liter of medium), and then TB / Amp medium (medium Inoculate 1% (v / v) in 12 g bacto-tryptone, 24 g yeast extract, 4 ml glycerol, 0.017 mole KH ₂ PO ₄ , 0.72 mole K ₂ HPO ₄ and 50 μg / ml empicillin) Shake culture at 37 ℃. When the OD ₆₀₀ reached 0.3, L-arabinose was added to induce the expression of GSTt / UK / PTH fusion protein so that the final concentration was 1% (W / V), followed by further shaking for 8 hours, followed by centrifugation. To obtain the cells.

Cells (4-6 g) obtained in the above were suspended in 8-12 ml of Tris buffer (50 mM Tris buffer containing 0.1 mM EDTA and 25% sucrose, pH 7.8) and then 0.2 mg / ml of lysozyme (lysozyme). ) Was added and reacted at 0 ° C. for 1.5 hours. Then, 20mM MgCl ₂ and 50μg / ml DNasel was added and reacted again for 1.5 hours. Then, 20 mM Tris buffer containing 13-20 ml of Tris buffer (0.2M NaCl and 1% deoxycholic acid, 1.6% Nonidet p-40, 2mM EDTA and 0.5mM dithiothreitol), pH 7.8) was added and the cells were lysed for 15 minutes with gentle stirring at 0 ° C.

The cell disruption solution was centrifuged at 12,000 rpm for 20 minutes to obtain an inclusion body, and then 100 ml of Tris buffer solution (20 mM Tris buffer solution containing 0.5% Triton X-100, 1 mM EDTA and 1 mM DTT), pH 7.8) in total four times. The inclusion body thus obtained was charged with 6.7 to 10 ml of Tris buffer solution (20 mM Tris buffer solution, pH7.8 containing 8 M urea and 20 μM DTT) and stirred slowly at 4 ° C. to denature the inclusion body. The denatured inclusion body was dialyzed in Tris buffer (20 mM Tris buffer solution containing 0.1 M NaCl and 20 μM DTT, pH7.8, hereinafter referred to as 'Buffer A') to refold the protein.

In order to determine what type of GSTt / UK / PTH fusion protein is expressed in E. coli, the samples were analyzed by SDS-PAGE during the electroencapsulation separation process (see FIG. 4). In FIG. 4, the first lane is cell lysate; The second lane is the supernatant of the cell lysate; The third to sixth lanes include washing liquid of the inclusion body; And the seventh lane each represent an enclosure. As shown in FIG. 4, the GSTt / UK / PTH fusion protein was expressed in Escherichia coli like the GSTt / FX / PTH fusion protein, and all of them were separated into inclusion bodies.

Example 4

Cleavage of Isolated GSTt / UK / PTH Fusion Proteins

The amount of GSTt / UK / PTH fusion protein isolated in Example 3 was determined, and then adjusted to a protein concentration of 4 mg / ml with buffer A. 1 μl of urokinase-containing solution (1 μg / μl) was added to 100 μ1 of GSTt / UK / PTH fusion protein solution prepared as described above so that the concentration ratio of GSTt / UK / PTH fusion protein and urokinase was 400: 1, and then at 25 ° C. The degree of cleavage by reaction for 1 hour was divided by SES-PAGE (see FIG. 5).

In FIG. 5, the odd lanes represent no addition of urokinase as a control, and the even lanes represent addition of urokinase; the first and second ray show the fusion protein of the cleavage site, Ile-Glu-Gly-Arg. Fusion proteins 3 and 4 are Ala-Pro-Gly-Arg, fusion proteins 5 and 6 are Gly-Pro-Gly-Arg, fusion proteins 7 and 8 are Gly-Phe-Gly-Arg, Fusion proteins 9 and 10 are Gly-Thr-Gly-Arg, fusion proteins 11 and 12 are Gly-Ile-Gly-Arg and fusion proteins 13 and 14 are Gly-Leu-Gly-Arg. , Lane M represents a molecular weight marker, respectively.

As shown in FIG. 5, -Pro-Gly-Arg cleavage sites of the 3 to 6 lanes, which were predicted to be efficiently cleaved by urokinase, were unexpectedly, Ile-Glu- which is a cleavage site of factor tenei. Since the Gly-Arg site was cut at a level similar to or lower than that of the urokinase, the electrical prediction site was found to be an inefficient cutting site of urokinase. On the other hand, it was found that the -Thr-Gly-Arg cleavage site of the ninth to tenth lanes was very efficiently cleaved by urokinase. In addition, the cutting efficiency by lokinase is significantly lower than that of -Thr-Gly-Arg cutting sites, but other cutting sites such as -Ile-Gly-Arg, -Len-Gly-Arg, and -Phe-Gly-Arg also have urokinase. It can be seen that to some extent by the. This means that the amino acid located at the P3 subsite of the urokinase substrate protein is an important factor influencing the substrate specificity of the urokinase, but at the same time, some degree of flexibility is allowed for the amino acid species that may be present at the P3 subsite.

On the other hand, in order to measure the cleavage efficiency of -Thr-Gly-Arg cleavage site by urokinase, the concentration ratio of GSTt / UK / PTH fusion protein and urokinase having cleavage site of Gly-Thr-Gly-Arg was 400: 1, The reactions were allowed to react at a constant temperature at 25 ° C. for 800: 1 and 1600: 1, and the degree of cleavage was analyzed by SDS-PAGE (see FIG. 6). In FIG. 6, the U lane is a fusion protein in which the amino acid sequence of the cleavage site is not Gly-Thr-gly-Arg without urokinase; The first to fourth lanes were reacted for 30 minutes, 1 hour, 2 hours, and 3 hours, respectively. The fifth lane had a concentration ratio of fusion protein and urokinase with an amino acid sequence of Ile-Glu-Gly-Arg of 400: 1. The reaction was carried out together at 25 ° C. for 3 hours; And, the sixth lane shows the result of reacting the fusion protein having the amino acid sequence of the cleavage site at Ile-Glu-Gly-Arg and the factor ten at 4 ° C. at 25 ° C. for 4 hours. As shown in FIG. 6, the fusion protein was almost 100% cleaved by urokinase in 2 hours even at the concentration ratio of the electrofusion protein and urokinase 1,600: 1. In addition, compared to the fact that only about 80% of the Ile-Glu-Arg sections were cleaved by the factor tenei for 4 hours at a concentration ratio of 400: 1 between the electrofusion protein and the factor teneigen, the Gly induced by urocanase The cutting efficiency per segment of -Thr-Gly-Arg cutting site was increased by at least 10 times compared to the hourly cutting efficiency of Ile-Glu-Gly-Arg cutting site by factor Tenei.

As described and demonstrated in detail above, the present invention expresses the specific amino acid sequence cleaved by urokinase between the target protein and the fusion partner of the fusion protein, and by cutting the expressed fusion protein with urokinase, Provided are methods for making proteins. In the present invention, when the amino acid sequence of the urokinase cleavage site located between the target protein and the fusion partner of the fusion protein is -X-Gly-Arg (X is Pro, Thr, Ile, Phe or Leu), the cleavage efficiency is excellent. It was found that the expression of the fusion protein including the cleavage site was excellent, and it was confirmed that by using the amino acid sequence of the urokinase cleavage site determined in the present invention, it was possible to economically and easily prepare a large amount of the protein from the fusion protein.

Claims (4)

A method of producing a target protein from a fusion protein comprising expressing a DNA sequence inferred from an aninoic acid sequence as follows between a target protein of a fusion protein and a fusion partner, and cleaving the expressed fusion protein with urokinase: -X-gly-arg Wherein X is Thr, Ile, Phe or Leu. Expressing the DNA sequence inferred from the sequence of -Thr-Gly-Arg between the target protein and the fusion partner of the fusion protein, and preparing the target protein from the fusion protein, comprising cutting the expressed fusion protein with urokinase. Way. The method of claim 1, wherein the DNA sequence located between the target protein of the fusion protein and the gene of the fusion partner is produced by site-specific mutations The method of claim 3, wherein the site-specific mutations are caused using mixed oligonucleotides such as -N ₁ N ₂ TGGAAGA In the above, N ₁ is A, C or T; And N 2 is C or T.