KR20030034136A - Fusion protein containing additional cationic amino acids and improvement of bio-operation by using same - Google Patents

Abstract

PURPOSE: A fusion protein containing additional cationic amino acids and an improvement method of bio-operation by using same fusion protein are provided, which cationic fusion gene can be used to enhance the purification, immobilization and refolding of a desired target protein. CONSTITUTION: The fusion protein comprises a target protein and more than two consecutive cationic amino acid residues, which are selected from lysine and arginine, fused to the target protein, wherein the number of the cationic amino acid residues is 6 to 15; the cationic amino acid residues are fused to the N-terminal or C-terminal of the target protein or located anywhere within the protein provided that they do not adversely affect the function of the protein; and the target protein is cyclodextrin glycosyltransferase (CGTase), lipase, disulfide-bond-promoting enzyme, disulfide bond isomerase, or proline isomerase.

Description

Fusion protein containing additional cationic amino acids and improvement of bio-operation by using same}

Genetic recombination techniques using microorganisms are widely used to produce medicinal and industrially useful proteins in large quantities, and according to the above method, proteins such as charge, solubility, size, hydrophobicity and affinity can be used to obtain proteins expressed in recombinant microorganisms. Various purification techniques using characteristics are used. At this time, the purification yield of the protein has a problem that is drastically dropped through a number of purification steps.

In addition, one of the biggest problems in mass production of the protein using the recombinant microorganism is that the produced protein does not have a correct tertiary structure, but forms an inert aggregate to form an inclusion body. Therefore, there is a need for a refolding process for recovering a recombinant protein expressed in an inclusion body form as an active protein. However, in such a refolding process, there is a disadvantage in that the efficiency is lowered due to reprecipitation due to hydrophobic bonds between the folding intermediates, which is particularly serious in the case of high concentration protein.

On the other hand, there are a variety of methods for producing an immobilized enzyme, the lowest cost immobilization method is a method of electrostatic adsorption on the carrier having the opposite charge by using the charge of the protein itself. However, this method has the disadvantage that the enzyme is separated from the carrier and is severely spilled into the solution.

Alternatively, by adding charged amino acids, the solubility, charge, or chemical properties of the protein may be modified to increase the efficiency of purification, refolding, and immobilization of the protein. For example, tags used for purification and identification of proteins include polyhistidine, FLAG peptide, strep-tag, polyaspartic acid, polyarginine, polyphenylalanine, polycysteine, and calmodulin- Binding peptides, green fluorescent peptides, and the like are used, some of which are commercially available. In particular, polyhistidine tag (Novagen, USA) is widely used at the laboratory level, but there is a problem in that an expensive carrier is required because an immobilized metal affinity chromatography (IMAC) method should be used.

In addition, a purification method using a fusion protein in which an anionic amino acid, a charged amino acid, is fused to a protein has been reported by Glatz et al., And cationic arginine acid is used for purification (Brewer, et al., Trends Biotechnol, 3, 119-122 (1985)), aspartic acid has been used for the immobilization of enzymes (Heng, et at., Biotechnology and Bioengineering, 44, 745-752 (1994); Suominen, et al., Biotechnol. Prog., 10, 237-245 (1994); and Enzyme Microb. Technol., 15, 593-600 (1993)), arginic acid has been used for immobilization and refolding (Stempfer, et al., Nature Biotec Hnology, 14, 451-484 (1996); Nature Biotechnology, 14, 329-334 (1996). However, these methods are not currently used for research as well as industrially. This is because, firstly, the method of purification is not simple and the degree of purification of the resulting protein is not as expected, and secondly, in some of the above methods, the cost may be increased by using a resin in which an expensive substance called heparin is bound. In addition, third, the application to biological processes is limited.

Accordingly, the present inventors continued to develop an amino acid fusion system in which purification, immobilization, and refolding processes are efficiently performed. As a result, cationic amino acids, arginine or lysine, may be mixed alone or in combination with the gene encoding the protein of interest. The present invention was completed by preparing a fusion protein in which two or more were fused.

Summary of the Invention

It is an object of the present invention to provide a fusion gene, a fusion protein encoded thereby, an expression vector comprising the gene and a transformant transformed with the vector, which can efficiently perform purification, immobilization, and refolding processes. .

Another object of the present invention is to provide a method for purifying, immobilizing and refolding the fusion protein.

According to one aspect of the invention there is provided a fusion protein in which two or more consecutive cationic amino acid residues selected from the group consisting of lysine and arginine are fused to a protein of interest.

According to another aspect of the present invention, there is provided a gene encoding the fusion protein and an expression vector pTCGTK6, pRCGTR6, pTCGTK10, pLPK10, pLPK12, pTDSBAK10, pTDSBR10, pTDSBCK10, pTDSBCR10, pTHPPIK10 or pTHPPIR10.

According to another aspect of the present invention, the transformant E. coli BL21 (DE3): pLysE: pTCGTK10 (Accession No .: KCTC 0842BP), E. coli BL21 (DE3): pLysE: pLPK10 E. coli BL21 (DE3): pLysE: pLPK12, E. coli BL21 (DE3): pLysE: pTCGTK6, E. coli BL21 (DE3): pLysE: pRCGTR6, E. coli BL21 (DE3): pLysE: pTDSBAK10, E. coli BL21 (DE3): pLysE: pTDSBR10, E. coli BL21 (DE3): pLysE: pTDSBCK10, E. coli BL21 (DE3): pLysE: pTDSBCR10, E. coli BL21 (DE3): pLysE: pTHPPIK10 or E. coli BL21 (DE3): pLysE: pTHPPIR10 is provided.

According to another aspect of the present invention there is provided a method for culturing the transformant and obtaining a cell extract from the culture medium in which the expression of the fusion protein is induced, and purifying the fusion protein using the ionic matrix.

According to another aspect of the present invention, after culturing the transformant and inducing the expression of the fusion protein, a method of purifying the fusion protein using an ionic matrix from a solubilized inclusion body is provided.

According to still another aspect of the present invention, there is provided a method for preparing an immobilized enzyme, characterized in that a cationic amino acid residue selected from the group consisting of lysine and arginine is bound to an enzyme by combining at least two consecutive fusion enzymes to the enzyme. do.

According to another aspect of the present invention there is provided a method of refolding an unfolded protein, characterized in that the defolded and unfolded fusion protein is adsorbed onto an ionic matrix followed by a refolding process.

The present invention provides a cationic fusion gene capable of increasing the purification, enzyme immobilization, and refolding efficiency of a protein, a fusion protein encoded thereby, an expression vector including the fusion gene, and transformation with the expression vector. And a method for purifying, immobilizing and refolding the fusion protein.

The above and other objects and features of the present invention will become apparent from the following description of the invention in conjunction with the accompanying drawings:

Figure 1 shows the manufacturing process of the expression vectors pTCGTK6, pTCGTK10 and pTCGTR6.

2a to 2d are chromatograms and analytical graphs of WT CGTase, CGTK6ase, CGTR6ase and CGTK10ase separated by chromatography using a cation exchange resin from recombinant E. coli cell extracts.

3 is a chromatogram and SDS-PAGE analysis of the eluted fusion protein after washing the recombinant E. coli cell extract with various salt concentrations.

Figure 4 is an analysis of SDS-PAGE purified by selectively adsorbing the fusion protein (CGTK10ase) to the ion exchange resin by adjusting the salt concentration of the recombinant E. coli cell extract.

Figure 5 shows the partition coefficient according to the salt concentration of the CGTK10ase protein of the present invention.

6 is a graph showing the titer of the enzyme adsorbed according to the salt concentration from the cation exchange column.

7 is a graph showing the change in pH dependence of the enzyme by amino acid fusion and immobilization using the same.

8 is a graph showing the change in pH stability of the enzyme by amino acid fusion and immobilization using the same.

9 is a graph showing the change in pH stability of enzymes by amino acid fusion and immobilization using the same.

10a to 10c are graphs showing the thermal stability of the fusion protein of the present invention and the protein immobilized thereto.

11A to 11 are graphs showing the productivity of cyclodextrin by immobilized enzyme.

Figure 12 shows the partition coefficient according to the salt concentration of the enzyme released by urea.

FIG. 13 is a graph comparing refolding efficiency of solution phase enzyme and immobilized enzyme according to Ca ++ concentration. FIG.

14 is a graph comparing the refolding efficiency of the solution phase enzyme according to the concentration of the salt.

15 is a graph comparing refolding efficiency of solution phase enzymes according to pH.

16 is a graph comparing the refolding efficiency of the immobilized enzyme according to the concentration of the salt.

17 is a graph comparing the refolding efficiency of the immobilized enzyme according to the pH.

18A and 18B are graphs comparing refolding efficiency of solution phase enzymes according to protein concentration.

19 is a graph comparing refolding efficiency of immobilized enzyme according to protein concentration.

Figure 20 is a chromatogram purified by selectively binding the fusion proteins CGTK10ase, LPK10ase expressed in the inclusion body form using a cation exchange resin.

FIG. 21 is a chromatogram of purified hPPIK10ase and hPPIR10ase using a cationic matrix.

22 is a graph showing the refolding efficiency of lipase, disulfide bond promoter, disulfide bond isomerase and proline isomerase according to protein concentration.

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

In the present invention, a fusion protein in which two or more, preferably six to fifteen, most preferably eight to twelve consecutive fusions of the cationic amino acid lysine or arginine are separately or mixed with the desired protein, respectively. can do. In the preparation of the fusion protein of the invention, the cationic amino acid can be inserted anywhere inside the enzyme, as long as it does not substantially affect the C-terminus of the protein, as well as the N-terminus or the functionality of the enzyme. Here, having no substantial effect on the functionality of the enzyme means that the enzyme retains at least 80%, preferably at least 95%, activity before the fusion of the cationic amino acid.

As the target protein used in the present invention, cyclodextrin glycosyltransferase (CGTase) derived from Bacillus mascerans, lipase derived from Archaeoglobus fulgidus, and lipase Binding promoter, disulfide isomerase, proline isomerase, and the like, but are not limited thereto. For example, the cationic fusion protein comprising CGTase may be in the form of CGTK10ase, a protein in which ten lysines are continuously fused to the C-terminus of a wild-type (WT) CGTase, and cationic including a lyphase. The fusion protein may be LPK10 and LPK12, in which 10 and 12 lysines are continuously fused at the C-terminus of the wild type lipase.

The present invention also provides a gene encoding the fusion protein. However, due to the degeneracy of the codons or taking into account the codons preferred in the organism in which the genes are to be expressed, the genes of the present invention may be modified in various ways without changing the amino acid sequence of the protein expressed from the coding region. This can be done. Thus, the present invention also encompasses polynucleotides having base sequences substantially identical to those genes.

The fusion protein of the present invention is an expression vector prepared by synthesizing a gene encoding the same, and inserting the same into a suitable vector, thereby transforming the transformant obtained by transforming the host, for example, yeast or E. coli, under an appropriate culture condition. Can be obtained by culturing. For example, in the case of the CGTase fusion protein, the 3 'of the WT CGTase gene was obtained using the vector pTCGT1 containing WT CGTase gene (Lee, KCP and BY Tao., Starch, 46, 67-74 (1994)). An expression vector pTCGTK10 comprising a fusion gene having 10 lysine codons linked to each end thereof is prepared, and transformed into E. coli (for example, E. coli BL21 (DE3)), and then cultured. Can be.

WT CGTase does not adsorb to cation exchange resins at pH 7.4 because the theoretical pI is about 5.0. However, CGTK10ase of the present invention, which is a fusion protein fused with cationic amino acids, is adsorbed by a cation exchange resin, and the purified protein CGTase can be purified using the same.

Therefore, in the present invention, a method for purifying or transforming a fusion protein comprising culturing the transformant and obtaining a cell extract from the culture medium inducing the expression of the fusion protein and purifying it using a cationic matrix. After culturing and inducing the expression of the fusion protein, there is provided a method for purifying a fusion protein comprising purification using a cationic matrix from a solubilized inclusion body. Cationic matrices used in the present invention include SP sepharose, S sepharose or CM sepharose, and SP sepharose is particularly preferable. However, they are not limited to these matrices and functional groups.

Emax of CGTK10ase is 580 mM when defining Namax concentration at the point of maximum activity during purification of protein by cation exchange chromatography, whereas Emax of CGTK6ase and CGTR6ase fused with 6 lysine and arginine codons is 345 mM and 430 mM, respectively. In addition, since WT CGTase does not adsorb at all on the cation exchange resin, Emax can be called 0. Emax represents the adsorption strength of the protein. A high Emax means that it is strongly adsorbed to the cation exchange resin. Therefore, the fusion protein with high Emax is not desorbed even by high salt concentration, and it can be used to separate the target protein from other intracellular proteins that are desorbed at relatively low salt concentration. The Emax of the fusion protein depends on the type and length of amino acids fused to the protein of interest. In the same length, the fusion of arginine is higher than that of lysine, and in the same species, the longer one is higher than the short. Also, fusion proteins with high Emax dissociate later. Most of the intracellular proteins dissociate at low salt concentrations, but there are proteins that are adsorbed at considerably high salt concentrations, for example 100-300 mM, which act as impurities in the final purified solution, resulting in poor purification. As such, the greater the Emax, the greater the purification fold. For example, the purification folds of CGTK6ase, CGTR6ase, and CGTK10ase are 2.38, 4.97, and 9.88, respectively.

In addition, the purification of the target protein can be further enhanced by pre-washing the column to which the target protein is adsorbed with a buffer to which 10 to 1000 mM of salt is added during cation exchange chromatography, which is a process of dissociating weakly adsorbed intracellular proteins. For example, when CGTK10ase is prewashed using 400 mM salt and analyzed using SDS-PAGE and densitometer, it shows a purity of 95% or more and is purified by affinity chromatography. Compared with pure protein and non-enzyme titer, the purity is about 98%. Through this method, a very high degree of purification protein can be obtained. In general, considering that the degree of purification of enzymes used industrially is about 90%, the present invention can be used directly without additional purification by using a simple purification method. Can produce enzymes.

Purification efficiency of the fusion protein can be adjusted not only in the dissociation step but also in the adsorption step, and the present inventors referred to this as 'selective binding', thereby increasing the recovery of the target protein and improving the purification. Intracellular proteins competitively bind to the protein of interest in the ion exchange resin. When the salt is added after the salt is added at a concentration of 100 mM to 500 mM, the intracellular protein is not only prevented from binding to the protein but also the yield of adsorption of the protein of interest and Purity can be increased dramatically.

In addition, the fusion protein of the present invention can be used for the preparation of the immobilized enzyme using electrostatic adsorption on the cation exchange resin when the target protein is an enzyme. For example, in the case of CGTase, even if the cationic amino acid is fused, the enzyme titer is maintained at 90% or more, and the immobilization of the same enzyme occurs by immobilization. The immobilized enzyme of the present invention can be prepared more easily than conventional methods, and can be used in the immobilized enzyme reactor while keeping the protein stable without being damaged or spilled during the immobilization process.

Fusion enzymes and their immobilized enzymes exhibit the same pH dependence as wild enzymes. In addition, the solution-type fusion enzyme shows the same pH stability as the wild type, but the pH stability becomes very high through immobilization.

In terms of thermal stability, the fusion enzyme was lower than that of the wild type, but immobilization of the fusion enzyme significantly improved the thermal stability. Therefore, by operating the enzyme reactor using the immobilized enzyme of the present invention it is possible to efficiently produce the desired product from the substrate of the enzyme.

The use of the fusion protein of the present invention can significantly increase the yield of the refolding and the performance of the process due to the added cationic amino acid, which is immobilized on the cation exchange resin after the protein released by the denaturant By removing the denaturant or lowering the concentration to induce refolding. In addition, the present invention can be successfully carried out while preventing the precipitation of the protein even when the high concentration of protein refolding.

Hereinafter, the present invention will be described in detail through examples. only. The following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Expression Vectors of Fusion Proteins Concatenated with Cationic Amino Acids

(1) Preparation of expression vector of CGTase fusion protein

The vector pTCGT1 (Lee, KCP and BY Tao., Starch, 46, 67) using the 5'-primer having the nucleotide sequence of SEQ ID NO: 1 and the 3'-primer having the nucleotide sequence of SEQ ID NO: 2 and containing the WT GCTase gene -74 (1994)) was used as a template to carry out a polymerase chain reaction (PCR). As a control, PCR was performed using a 3'-primer having a nucleotide sequence of SEQ ID NO: 3 or 4. Using 1 mM MgCl ₂ , 30 cycles of denaturation reaction at 95 ° C. for 1 minute, annealing reaction at 48 ° C. for 2 minutes and polymerization reaction at 72 ° C. for 3 minutes, and further polymerization at 72 ° C. for 5 minutes The reaction was carried out. The initial denaturation reaction was carried out for 5 minutes at 95 ℃. The DNA fragments thus obtained were separated by agarose gel electrophoresis to confirm their size and then treated with restriction enzymes BamHI (NEB, England) and SalI (NEB, England).

On the other hand, plasmid pTCGTK10 (SEQ ID NO: 5) containing a gene encoding 10 lysine by treating the vector pET21a (Novagen, USA) with the same restriction enzyme and ligation with the restriction enzyme-treated fragment, as a control Plasmids pTCGTK6 (SEQ ID NO: 7) and pTCGTR6 (SEQ ID NO: 9) containing genes encoding six consecutive lysines and arginine, respectively, were obtained. Figure 1 shows the manufacturing process of the expression vectors pTCGTK6, pTCGTR6 and pTCGTK10.

(2) Preparation of Expression Vector of Lypha Phase Fusion Protein

A polymerase chain reaction (PCR) was carried out using a 5'-primer having a nucleotide sequence of SEQ ID NO: 11 and a 3'-primer having a nucleotide sequence of SEQ ID NO: 12 or 13, and pELP-1 as a template. pELP-1 is an expression vector in which the phase gene of Achaeoglobus fulgidus (ATCC 49558D) is inserted into the restriction enzymes NdeI and BamHI of pET3a vector (Novagen, U.S.A.). The polymerase chain reaction was carried out in the same manner as in (1), and the obtained DNA fragment was treated with restriction enzymes NdeI (NEB, England) and BamHI (NEB, England). Vector pET3a (Novagen, USA) was treated with the same restriction enzyme as above and ligated with the restriction enzyme treated fragments to obtain plasmids pLPK10 and pLPK12 comprising genes encoding consecutive 10 and 12 lysines, respectively.

(3) Preparation of expression vector of disulfide bond promoter, disulfide isomerase and proline isomerase fusion protein

5'-primer having the nucleotide sequence of SEQ ID NO: 14 and 3'-primer having the nucleotide sequence of SEQ ID NO: 15 were mixed, followed by annealing at room temperature, and then polynucleotide kinase (NEB, USA). Treatment was carried out at 37 ° C. for 1 hour. pET29-b (Novagen, U.S.A.) was treated with XhoI and BamHI restriction enzymes and then ligated with the annealed DNA fragment to obtain pETK10. On the other hand, pETR10 was obtained by the same method as above using the 5'-primer having the nucleotide sequence of SEQ ID NO: 16 and the 3'-primer having the nucleotide sequence of SEQ ID NO: 17. A polymerase chain reaction was carried out using cDNA of human proline isomerase (Accession Number: ATCC 78809) as a template and a nucleotide sequence of SEQ ID NOs: 18 and 19 using a primer, and the obtained DNA fragment was subjected to restriction enzyme NdeI (NEB, England) and BamHI (NEB, England). In addition, E. coli chromosomes were used as templates to obtain disulfide bond promoters, and polymerase chain reaction was carried out using primers having the nucleotide sequences of SEQ ID NOs: 20 and 21, and subjected to the same restriction enzyme treatment. In addition, in order to obtain disulfide isomerase, E. coli chromosome was used as a template, and primers having the nucleotide sequences of SEQ ID NOs: 22 and 23 were used to perform a polymerase chain reaction and the same restriction enzyme was treated as above.

Subsequently, the prepared vectors pETK10 and pETR10 were treated with the same restriction enzyme as described above and linked to the restriction enzyme-treated fragments, respectively, to include plasmids pDSBAK10, pDSBCK10, and pHPPIK10, each of which contains a sequence of 10 lysine or arginine sequences. , pDSBAR10, pDSBCR10 and pHPPIR10 were obtained.

Example 2 Purification of CGTase Fusion Protein Using Ion Exchange Chromatography

E. coli BL21 (DE3): pLysE (Novagen, USA) was transformed with the expression vector pTCGTK10 prepared in Example 1 (1), and then transformed colonies were selected from ampicillin-containing plates. The cell line E. coli BL21 (DE3): pLysE: pTCGTK10 was obtained. It was deposited with KCTC (Korean Collection for Type Cultures) on July 21, 2000 as Accession No. KCTC 0842BP.

E. coli BL21 (DE3) to produce WT GCTase as the transformant and the control group: pLysE: pTCGT1 and Example 1 (1) transformed with the expression vector and pTCGTK6 pTCGTR6 obtained in E. coli BL21 (DE3 ): pLysE: pTCGTK6 and E. coli BL21 (DE3): pLysE: pTCGTR6 were inoculated in 5 ml of LB medium (including 50 mg / l of ampicillin) and incubated overnight at 37 ° C, and 1 ml of this was 2 g / l. 100 ml of LB medium containing glucose was inoculated again and incubated at 30 ° C. After 6 hours of culture, when glucose was consumed, 0.5 mM of IPTG and 5 mM of CaCl ₂ were added to induce protein expression. Cells harvested by centrifugation were suspended in 50 mM phosphate buffer (pH 7.4) and ground using a French presser (French pressure, Aminco, USA).

The supernatant containing only the soluble cell extract was recovered using a centrifuge and analyzed by ion exchange chromatography. That is, 5 ml of SP-Sepharose (Pharmacia, Sweden) cation exchange resin was charged into an XK16 column (Pharmacia, Sweden), and then the supernatant was loaded and phosphate buffer (pH 7.4) was used as a mobile phase for 20 minutes ( 4 volumes of resin) was flowed at a flow rate of 1 ml / min. The gradient was then run over 20 minutes using a solution with 1 M NaCl added to the same mobile phase. Afterwards, 1 M NaCl was continuously flowed for 20 minutes. Absorbance, conductivity, and enzyme titer at 280 nm were observed during operation.

2 is a chromatogram and analytical graph separated from a recombinant E. coli cell extract using a cation exchange resin, wherein a, b, c, and d are chromatograms for WT CGTase, CGTK6ase, CGTR6ase, and CGTK10ase, respectively; (----) is the 280 nm UV curve, (-·-) is the gradient, (-··-) is the conductivity and the bar represents the enzyme titer. As shown here, most of the proteins were not adsorbed to the resin when the pH 7.4 buffer was used, and the WT CGTase was also negatively charged at the designated pH because the isoelectric point was about 5.0 (Fig. 3). A). B, C and D of FIG. 3 showed almost similar tendency for most separation characteristics except the titer of the enzyme. The pKa of lysine is 10.0 and the pKa of arginine is 12.0. When comparing the same length of lysine tail and arginine tail, the adsorption intensity of the fusion protein with arginine tail was higher. The intensity of CGTK10ase binding was significantly higher than that of control CGTR6ase and CGTK6ase. The results are shown in Table 1 below.

CGTase CGTK6ase CGTK10ase CGTR6ase Input protein (mg) 1.04 1.18 1.00 1.00 Input Enzyme Titer (U) 17.26 5.02 9.76 7.48 Non-enzymatic titer of injected protein (U / mg) 17.26 5.92 9.76 7.48 Total titer recovered (U) 0 2.22 4.92 1.45 Total recovered titer yield (%) 0 44.2 50.0 19.4 Nonenzymatic titer of recovered protein at E _max (U / ml) 0 14.1 96.4 37.2 Tablet drainage at E _max 0 2.38 9.88 4.97

Example 3 Purification of Purity of Fusion Proteins Through Pre-Washing

As in Example 2, a recombinant E. coli cell extract containing WT CGTase, CGTK6ase, CGTR6ase, and CGTK10ase was obtained, and cation exchange chromatography was performed as follows. Cell extracts were loaded on a column and adsorbed, followed by pre-washing with phosphate buffers with salt (NaCl) concentrations of 0, 150, 200, 300 and 400 mM, respectively, followed by elution. 12% SDS-PAGE was performed using the eluate, and the gel was Coomassie stained.

3 is an elution graph and SDS-PAGE analysis showing the results of pre-washing the recombinant E. coli cell extract, A is CGTKase to 150 mM NaCl, B is WT CGTase to 200 mM NaCl, C is CGTR6ase to 200 mM NaCl D is the pre-wash of CGTK10ase with 400 mM NaCl, and S is the standard molecular weight protein. WT CGTase did not adsorb even in salt solution with zero salt concentration. At this time, it was confirmed that a significant amount of intracellular proteins remained unadsorbed without being washed, and even when the salt concentration was increased to 200 mM, the amount was reduced but the intracellular proteins remained without being completely removed (FIG. 3B). . In the case of CGTK6ase, CGTK6ase remained adsorbed when pre-washed with buffer only, but it was desorbed when prewashed with salt concentration of 150 mM, and there were still intracellular proteins that were still strongly adsorbed and not dissociated. It was found (A in FIG. 3). Although CGTR6ase, which has a relatively strong binding to the cation exchange resin, was desorbed at the salt concentration of 300 mM, it was adsorbed at 200 mM, and it can be seen that a large part of the intracellular protein was not removed even at this time. (FIG. 3C). In the case of CGTK10ase of the present invention, even when pre-washed at a salt concentration of 400 mM remained undesorbed, most of the intracellular proteins other than the fusion protein were removed (D in FIG. 3). Some strongly adsorbed intracellular proteins could not be removed by 300 mM pre-washing, but most of them could be removed by washing with 400 mM salt concentration. These observations also show the same trend in the protein curves at the absorbance measurements at 280 nm. The higher the concentration of pre-washed salt, the more desorbed to 1 M salt concentration and the lower the recovered protein concentration. Intracellular proteins were removed by pre-washing.

(Example 4) Purification degree and purification yield improvement through selective adsorption

To the recombinant E. coli cell extract containing CGTK10ase obtained in Example 2, salts were added at concentrations of 0, 100, 200, 300 and 400 mM, followed by SP-sepharose cation exchange packed in a Poly-prep column (Biorad, USA). After adsorbing to the resin, and washed with the same concentration of salt solution. Proteins adsorbed with 1 M salt solution were analyzed by SDS-PAGE to obtain a zymogram as shown in FIG. 4. 4 is an SDS-PAGE analysis photograph selectively selecting the ion exchange reaction of the cell extract during the purification of CGTK10ase expressed in recombinant Escherichia coli, wherein S is a standard molecular weight protein and A is an affinity chromatography (Sundberg, L. And Porath J., J. of Chromatogr., 90, 87-98 (1974)), and B was purified by 400 mM salt solution pre-wash and cation exchange chromatography according to the method of Example 3. Protein, C, D, E, F and G are proteins purified by 1M salt solution after adsorption of cell extract supernatant with 0, 100, 200, 300 and 400 mM salt solution to cation exchange resin, respectively. to be. As shown here, as the adsorption salt concentration increases from 0 to 300 mM, the adsorption of intracellular proteins gradually decreases, and the intracellular proteins compete with the fusion proteins for ion exchange resins, so their adsorption prevention prevents the recovery of fusion proteins. By increasing it, more fusion proteins such as D, E, F and G can be recovered and purification efficiency can be increased.

Example 5 Enzyme Immobilization Via Cationic Amino Acid Fusion

(Step 1)

Partition coefficients were measured at various salt concentrations in the following manner. The pure purified CGTK10ase solution was equilibrated with an ion concentration of 0 to 500 mM (20 mM phosphate buffer, pH 7.0), followed by mixing the equilibrated resin (SP-Sepharose, Pharmacia, Sweden) at the same salt concentration. Was immobilized. The partition coefficient was calculated by measuring the amount of protein adsorbed and the amount of protein in the solution, and the concentration of the adsorbed protein was q and the concentration of protein in the solution was p.

Figure 5 shows the partition coefficient according to the salt concentration of the CGTK10ase protein of the present invention. As shown here, the partition coefficient at salt concentration of 0 to 300 mM was very well immobilized to about 0.95, and it was confirmed that the fusion enzyme CGTK10ase was immobilized by a noncovalent method rather than a covalent bond.

(Step 2)

In order to investigate the outflow of CGTK10ase immobilized on a cation exchange resin in a buffer solution of 0-1 M salt concentration (20 mM phosphate buffer, pH 7.0), the titer of the enzyme in the outflow solution after equilibration was measured. After binding the fusion enzyme to the cation exchange resin, the unbound fusion enzyme was washed and removed, and then incubated with light mixing for 10 hours in a 10 volume salt solution. In general, the adsorption and desorption reactions of proteins are known to reach equilibrium within a few minutes, so 4 hours of incubation was considered to be sufficient equilibrium time. The efflux curve of the fusion enzyme according to salt concentration is shown in FIG. 6 when the sum of the titer of the enzyme which has been released into the solution during the cultivation and the titer of the enzyme after desorption with 1 M NaCl is 100. In Figure 6 the black spot is the enzyme that has not leaked and the white point is the titer of the leaked enzyme. As shown in the graph, there was no leakage of immobilized enzyme at the salt concentration of 100 mM or less, and even at a concentration of 300 mM, more than 90% of the enzyme was immobilized and did not leak.

Example 6 Determination of the dependence and stability of the enzyme on the pH

To determine the pH dependence of the fusion enzyme and its immobilized enzyme, 0.05 ml of WT CGTase, unimmobilized CGTK10ase and immobilized CGTK10ase were added to 1.45 ml of phosphate buffer adjusted to pH 4-9, respectively, and the enzyme titer was determined. After the measurement, the point showing the highest titer was 100 and the relative value was indicated. As shown in FIG. 7, the enzyme in the liquid phase showed almost the same pattern of WT CGTase and CGTK10ase, and the immobilized enzyme also showed almost the same enzyme dependence curve at pH 6 and above. This may be due to the leakage of the enzyme due to the weak electrostatic binding force. Therefore, the enzyme and the immobilized enzyme fused with the cationic amino acid at the C-terminus of CGTase can be confirmed that the dependence on pH does not change compared to wild enzyme.

On the other hand, in order to measure the stability of the fusion enzyme and its immobilized enzyme pH, WT CGTase, unimmobilized CGTK10ase and immobilized CGTK10ase were placed in a phosphate buffer adjusted to pH 4 to 10 and left at 50 ° C. for 1 hour. The enzyme titer was then measured. The point showing the highest titer is 100 and the rest is expressed as a relative ratio to it. As can be seen in Figure 8, WT CGTase and CGTK10ase showed almost the same profile in pH stability, it was confirmed that the immobilized enzyme is stable in a wider pH range.

If the inactivation of the enzyme is made exponentially it can be expressed as Equation 2 below. Where A is the remaining enzyme titer, t is time, and k is the inactivation constant.

WT CGTase and CGTK10ase in the liquid phase showed almost the same value for the applied pH range, but immobilized CGTK10ase showed more reduced inactivation constant. The inactivation rate of the enzyme can be determined by measuring the titer of the remaining enzyme at time intervals while culturing the enzyme in the same manner as described above. The enzyme was exponentially inactivated as given above and the rate constant at this time is shown in FIG. 9. As a result, the fusion of cationic amino acids does not affect the pH stability of the enzyme, and it can be seen that an enzyme more stable to pH can be obtained through immobilization of the fused enzyme.

Example 8 Measurement of Thermal Stability of Immobilized Enzyme

In order to investigate the stability of the enzyme with temperature, the temperature was adjusted to 50 ° C. and 25 ° C. in 20 mM phosphate buffer (pH 7.0) to measure the degree of decrease in the titer of the enzyme with time, and the results are shown in FIG. 10. 10a and 10b are graphs showing the thermal stability of the fusion protein of the present invention and the protein immobilized thereon at 50 ° C. and 25 ° C., respectively. As shown here, the thermal stability of CGTK10ase is slightly decreased compared to the solution-phase WT CGTase. Immobilized CGTK10ase showed the same or slightly improved thermal stability as WT CGTase.

CGTase also requires Ca salts to maintain its structure. Therefore, after adding CaCl ₂ to 5 mM, the buffer was measured for the thermal stability of the enzyme at a temperature of 50 ° C. using 20 mM MOPS (3- [N-Morpholino] propanesulfonic acid, Sigma, USA) (pH 7.0). Shown in As shown here, the overall thermal stability of the enzyme was greatly increased, and it was also confirmed that the thermal stability of the enzyme was significantly improved by immobilization.

Example 9 Measurement of Operational Stability of Immobilized Enzyme

Immobilized enzyme was made by mixing CGTK10ase solution equilibrated with 10 mM phosphate buffer (pH 6.0) with SP-Sepharose equilibrated with the same buffer. Unimmobilized enzymes were washed off and placed in an XK16 column (Pharmacia, Sweden) to create a packed reactor. The column was maintained at 25 ° C. using a water jacket, and the peristaltic pump was added at a flow rate of 0.5 ml / min to a reaction solution containing 10 g / l of water-soluble starch in a phosphate buffer containing CaCl ₂ at 5 mM. Injection into the column continuously. Samples were taken once a day for 15 days and placed at −20 ° C. The resulting cyclodextrins (CD) were analyzed using thin film liquid chromatography (TLC) as follows. Cyclodextrins (CD) include α-CD consisting of six glucose molecules and β-CD and γ-CD consisting of seven and eight molecules of glucose, respectively.

TLC plates (KF5, Whatman, USA) were activated for 1 to 2 hours in an oven at 110 ° C. before use, and then diluted 1-2 samples to be included in the concentration range of the standard, spotting 1-2 μl. The standard was 0.005 to 0.05% glucose, maltose, maltotriose and each CD spotted together during sample analysis to obtain a standard graph to be converted for each plate. The plate to which the sample was applied was sufficiently dried in a dryer and developed twice in a chamber with a solvent (nitromethane: water: n-propanol, 2: 1.5: 4, v / v). After each development, it was developed by placing in an oven for 10 minutes until there was no smell of solvent. Methanol: H 2 SO 4 (including 95: 5, v / v, 3 g / L a-naphthol) solution was applied to the developed plate, dried well, and placed in a 110 ° C. oven for 10 minutes to develop. The color spots were converted into concentrations using a densitometer (GS-700, Biorad, USA) and a quantitative calculation program (Molecular Analyst, Biorad, USA). FIG. 11 is a graph showing the productivity of cyclodextrin (CD) by immobilized enzymes, where A, B, C and D represent α-CD, β-CD, γ-CD and total CD, respectively. As shown here, it was confirmed that the cyclodextrin is stably produced using the immobilized enzyme, it was confirmed that there is no change in the various CD content.

As shown in Examples 5 to 9, the immobilized enzyme could be prepared simply and efficiently by fusing cationic amino acid to the enzyme, and the immobilized enzyme thus produced was used to produce the desired product successfully.

Example 10 Immobilization of Denatured Protein

Whether or not the protein denatured by urea (fixed) is immobilized on the cation exchange resin was investigated in the following manner. In a 20 mM MOPS (3- [N-Morpholino] propanesulfonic acid, Sigma, USA) buffer containing 9 M urea (pH 7.0), NaCl was added at 0 to 400 mM, CGTK10ase was mixed and denatured for 4 hours. Immobilized in SP-Sepharose equilibrated in the same solution. The ratio of the volume of the resin and the volume of the protein solution was 1: 2, and the mixed solution was immobilized by reacting with a light mixture at room temperature for 2 hours. The solution after the resin was settled was recovered and the protein concentration was measured. This concentration was referred to as p. The sunken resin was thoroughly washed with the same solution and then all the attached proteins were dissociated by adding about 17 times the volume of the resin with 20 mM phosphate solution (pH 7.0) containing 1 M NaCl and 9 M urea. At this time, the concentration of the protein in the solution was measured, and the concentration of the protein that was immobilized on the resin was referred to as q, and the partition coefficient was measured according to Equation 1 shown in Example 5. The results are shown in FIG. 12, and as shown here, the distribution coefficient was almost constant at 0.9 to 100 mM at salt concentrations of 0 to 100 mM, and began to decrease rapidly above that value, approaching 0 at 400 mM salt concentrations. That is, the denatured fusion enzyme was also confirmed that it is well immobilized like a natural enzyme.

Example 11 Refolding Unlocked Protein

(1) refolding process

SP-separ equilibrated under the same conditions to denature CGTK10ase, which was left at room temperature for 4 hours or more in 20 mM MOPS (3- [N-Morpholino] propanesulfonic acid, Sigma, USA) buffer containing 9 M urea (pH 7.0). Immobilized by mixing with Rose, and washing off all unimmobilized protein. CGTK10ase and the immobilized enzyme in solution were placed in refolding buffer (20 mM MOPS buffer, pH 7.0) at 50 mg / ml and 1 mg / ml concentrations. After dilution, the concentrations of urea were all 0.45 M, and the diluted solution was left at 15 ° C. for about 16 hours to measure the titer of the enzyme. Immobilized enzyme was desorbed with 1 M NaCl and the protein concentration was measured. The enzyme activity divided by the protein concentration was called specific enzyme activity, and the refolding yield was calculated by comparing with the nonenzyme titer (100%) of the natural enzyme.

(2) Refolding yield of CGTase according to the concentration of Ca salt

CaCl ₂ was added to the refold buffer at various concentrations and refolded in the same manner as in (1). As shown in Figure 13 it can be seen that the refolding yield is increased by the addition of the Ca salt and Ca salt is essential for the formation of the tertiary structure of CGTase.

(3) Refolding yield of CGTase according to ionic strength and pH

Refolding was carried out using a mixture of WT CGTase, CGTK10ase and SP-Sepharose resin and WT CGTase in an unimmobilized solution, and adding 0-400 mM NaCl to the refolding buffer (1). WT CGTase was used as a control to examine the effect on the refolding of the resin due to other protein sites inside the CGTase other than the lysine fusion site since the resin was not added to the resin and immobilized. As can be seen in Figure 14, WT CGTase was not significantly affected by the NaCl concentration over the whole period, even when the resin was added, there was a slight decrease in yield, but the effect was insignificant. In the case of CGTK10ase, the refolding yield was higher than that of WT CGTase, and it was significantly affected by NaCl concentration. As the NaCl concentration increased, the refolding yield increased, and the refolding yield in 400 mM NaCl was approximately 40%, which was about twice that of WT CGTase. The refolding yield of the solution phase CGTK10ase is thought to be due to the reduced precipitation due to the intermolecular repulsion caused by the strong charge given to the fusion protein. As a result, the refolding yield of the fusion enzyme was improved when the wild enzyme and the fusion enzyme were refolded in solution. Meanwhile, refolding was carried out as in (1) by using a solution phase WT CGTase, CGTK10ase and a mixture of resin and WT CGTase and adjusting the pH of the refolding buffer to 6 to 8.5. The isoelectric point (pI) of CGTase is about 5.0, and the net charge becomes more negatively charged at higher pH. By observing the refolding yield of the WT CGTase to which the resin was added at the same time, it can be seen that the charge inside the CGTase affects the refolding yield through interaction with the resin, as shown in FIG. There was no significant effect on refolding of WT CGTase. CGTK10ase also showed higher refold yield than WT CGTase.

(4) Yield yield of immobilized CGTK10ase according to ionic strength and pH

Using the immobilized CGTK10ase, refolding was carried out as in (1) by adding 10-400 mM NaCl to the refolding buffer.

In FIG. 16, the refolding yield of immobilized CGTK10ase was shown according to NaCl concentration. The refolding yields of the non-enzymatic titers of refolding enzyme were 100% at all NaCl concentrations. However, as can be seen from the black dots in Figure 16, the higher the salt concentration, the more the protein is dissociated to some extent and the overall recovery is also lowered.

In addition, using the immobilized CGTK10ase, the pH of the refold buffer was adjusted to 6 to 8.5, and refolding was carried out as in (1). 17 is a graph showing the refolding yield of immobilized CGTK10ase versus pH, and the overall refolding yield was much higher than that of WT CGTase and solution phase CGTK10ase of (2). The amount of remaining protein was almost constant between pH 6.0 and 8.5, but in the case of refolding yield, it was greatly influenced by pH, and the refolding yield decreased below pH 7.0, where the amount of positively charged sites in the protein increased. This suggests that when refolding on a cation exchange resin with a strong negative charge, it is important to minimize the amount of positive charge in the protein and minimize the attraction between the resin and the charged site inside the protein.

(5) Refolding efficiency of enzyme according to protein concentration

Refolding was carried out as in (1) by adjusting the concentration in the refolding buffer of WT CGTase, solution phase CGTK10ase and immobilized CGTK10ase to 0.004-8 mg / ml.

18 and 19 are graphs showing refolding efficiencies of enzymes according to protein concentrations of solution-phase enzymes and immobilized enzymes, respectively. FIG. 18A is a graph showing the activity of recovered enzymes, and FIG. 18B shows the recovery of non-enzyme titers. It is a graph. As shown here, the yield of the enzyme in solution decreased exponentially with increasing concentration of refolding. This is because reaggregation occurs between refolded intermediates or denatured proteins, and the higher the concentration, the larger the reaggregated portion. At protein concentrations above 0.1 mg / ml, only 4% of WT CGTase and 7% of CGTK10ase were refolded. However, as shown in FIG. 19, the fusion enzyme immobilized by the lysine fusion site was 100% refolded even at a protein concentration of 8 mg / ml. Compared with 0.1 mg / ml WT CGTase yielding a refolding yield of about 4%, the overall process performance was approximately 2,000, with a 25 times refolding yield at 80 times protein concentration through immobilized refolding using a lysine fusion site. It can be said that the ship has improved.

As described above, the yield of the enzyme in solution was not good even though the refolding at a very low protein concentration, the immobilized enzyme was very high despite the very high protein concentration. This is because a solution of the enzyme in the refolding process, a large portion of the protein re-aggregation between the refolded intermediates can not be properly folded, but in the case of immobilized enzyme re-aggregation between the refolded intermediates was prevented.

(Example 12) Purification of CGTK10ase expressed in lysine fused phase and inclusion body form

E. coli BL21 (DE3): pLysE (obtained from Novagen, USA), respectively, was transformed with the expression vectors pLPK10 and pLPK12 prepared in Example 1 (2), and then transformed into colonies containing ampicillin. Were screened.

E. coli BL21 (DE3): pLysE: pLGT, which produces CGTK10ase Cell line E. coli BL21 (DE3): pLysE: pELP-1, which produces pTCGTK10 and WT phases and the transformed cell line E. coli BL21 (DE3): pLysE: pLPK10, E. coli BL21 (DE3): pLysE: pLPK12 were inoculated in 5 ml of LB medium (including 50 mg / L of ampicillin). This was incubated overnight at 37 ° C., and then 1 ml of which was inoculated again in 100 ml of LB medium containing 2 g / L of glucose and incubated at 37 ° C. At 4 hours of culture, when glucose was consumed, 1 mM of IPTG was added to induce the expression of the protein. Cells harvested by centrifugation were suspended in 50 mM phosphate buffer (pH 7.4) and ground using a French presser (French pressure, Aminco, USA). Since the expressed phase was mostly made of an inclusion body, the supernatant, a soluble cell extract, was removed using a centrifuge, and the precipitate was dissolved in 9 M urea (20 mM MOPS buffer, pH 7.0). The solubilized protein was purified by the selective binding method described in Example 4 and analyzed by SDS-PAGE is shown in FIG. 21. In FIG. 20, S is a standard molecular weight protein and lanes 1 and 5 were obtained from the inclusion bodies recovered by E. coli BL21 (DE3): pLysE: pTCGTK10 and E. coli BL21 (DE3): pLysE: pLPK10, respectively. It is a sample. Lanes 2, 3, 4 and lanes 6, 7, 8 contained the inclusions of E. coli BL21 (DE3): pLysE: pTCGTK10 and E. coli BL21 (DE3): pLysE: pLPK10 at 300, 350 and 400 mM NaCl, respectively. After binding selectively, the enzyme solution was washed with the same concentration of salt solution and desorbed with 1 M NaCl. It can be seen that proteins denatured by urea can also be successfully purified using the lysine fusion site, and the purification is also very high.

(Example 13) Purification of disulfide bond promoter, disulfide bond isomer and proline isomerase to which 10 lysine and arginine were added

A plate containing ampicillin after transforming E. coli BL21 (DE3): pLysE (Novagen, USA) with the expression vectors pDSBAK10, pDSBCK10, pHPPIK10, pDSBAR10, pDSBCR10 and pHPPIR10 prepared in Example 1, respectively. E. coli BL21 (DE3): pLysE: pDSBAK10, E. coli BL21 (DE3): pLysE: pDSBCK10, E. coli BL21 (DE3): pLysE: pHPPIK10, E. coli BL21 (DE3): pLysE: pDSBAR10, E. coli BL21 (DE3): pLysE: pDSBCR10 and E. coli BL21 (DE3): pLysE: pHPPIR10.

The same method as in Example 3 was carried out to purify the disulfide bond promoter, disulfide bond isomerase and proline isomerase to which 10 lysine and arginine were added, and the results are shown in FIG. 21. As shown here, as in CGTase, these enzymes were successfully purified using a cationic amino acid fusion system.

Example 14 Solid Phase Refolding of Lyphais, Disulfide Bond Enzyme, Disulfide Isomerase and Proline Isomerase Using Cationic Amino Acid Fusion System

In the same manner as in Example 11, the solid phase refolding of lipase, disulfide bond promoter, disulfide bond isomerase and proline isomerase was performed, and the results are shown in FIG. 22. As seen here, even at high protein concentrations above 1 mg / mL, refolding of the protein was possible without precipitation, which is superior in yield compared to the conventional solution phase refolding, and can greatly reduce the volume of the process. The efficiency of the process could be dramatically increased.

Claims (17)

Fusion protein in which two or more consecutive cationic amino acid residues selected from the group consisting of lysine and arginine are fused to a protein of interest The fusion protein according to claim 1, wherein the cationic amino acid residues are fused to 6-15. 2. The fusion protein of claim 1, wherein the cationic amino acid residue is present at the C-terminus, N-terminus or site of the protein of interest without substantially affecting the function of the protein. The fusion protein according to claim 1, wherein the target protein is cyclodextrin glycosyltransferase, lipase, disulfide isomerase, disulfide bond promoter or proline isomerase. Gene encoding the fusion protein of any one of claims 1 to 4. Expression vector comprising the gene of claim 5 The expression vector pTCGTK6, pRCGTR6, pTCGTK10, pLPK10, pLPK12, pTDSBAK10, pTDSBR10, pTDSBCK10, pTDSBCR10, pTHPPIK10 or pTHPPIR10 A transformant transformed with the expression vector of claim 6 or 7. The method of claim 8, wherein the transformants E. coli BL21 (DE3): pLysE: pTCGTK10 (Accession No .: KCTC 0842BP), E. coli BL21 (DE3): pLysE: pLPK10 and E. coli BL21 (DE3): pLysE: pLPK12, E. coli BL21 (DE3): pLysE: pTCGTK6, E. coli BL21 (DE3): pLysE: pRCGTR6, E. coli BL21 (DE3): pLysE: pTDSBAK10, E. coli BL21 (DE3): pLysE: pTDSBR10, E. coli BL21 (DE3): pLysE: pTDSBCK10, E. coli BL21 (DE3): pLysE: pTDSBCR10, E. coli BL21 (DE3): pLysE: pTHPPIK10 or E. coli BL21 (DE3): pLysE: pTHPPIR10 A method for purifying a fusion protein comprising culturing the transformant of claim 8 and obtaining a cell extract from the culture medium inducing the expression of the fusion protein and purifying it using an ionic matrix. The method of claim 10, further comprising pre-washing with a buffer having a salt concentration of 0 to 1000 mM. The method of claim 10, wherein the salt concentration is adjusted to 100 to 500 mM by adding salt to the cell extract. A method for purifying a fusion protein comprising culturing the transformant of claim 8 and inducing the expression of the fusion protein, and then purifying the solubilized inclusion body using an ionic matrix. Cationic amino acid residue selected from the group consisting of lysine and arginine to the ionic matrix is a method for producing an immobilized enzyme, characterized in that to combine the fusion enzyme fused at least two consecutively to the enzyme Immobilized Enzyme Prepared by the Method of Claim 14 An enzyme reaction method characterized in that the immobilized enzyme of claim 15 is reacted with a substrate. Method for refolding unfolded protein, characterized in that the defolded (unfolded) fusion protein of claim 1 is adsorbed on the ionic matrix and then refolded