KR20000066106A - Process for Preparing Immobilized Urokinase by Semi-covalent Binding - Google Patents

Abstract

PURPOSE: Provided is a preparation method of immobilized urokinase for obtaining a target protein economically from recombinant protein which is expressed as fusion protein type being cleaved by urokinase. CONSTITUTION: A method for preparation of immobilized urokinase to adsorption resin by semi-covalent bond comprises the following steps: adding 0.05-1.0 mg of urokinase per 1 ml of hydroxylapatite resin suspended in buffer solution; stirring them; adding bovine serum albumin to the produced hydroxylapatite resin wherein urokinase is adsorbed; stirring them and followed by washing them with buffer solution; adding 2-30 micro/ml of glutaraldehyde to the produced hydroxylapatite resin where urokinase and bovine serum albumin are adsorbed and stirring them, followed by washing them with buffer solution. Wherein the buffer solution is 20-50 mM of Tris.HCl and of which pH is 7.0-90 and stirring is proceeding at 1-10°C for 10-100 minutes.

Description

Process for Preparing Immobilized Urokinase by Semi-covalent Binding}

The present invention relates to a method for producing immobilized urokinase. More specifically, the present invention relates to a method for preparing immobilized urokinase for economically obtaining a target protein from a recombinant protein expressed in the form of a fusion protein that can be cleaved by urokinase.

With the development of genetic engineering, methods for producing necessary proteins using recombinant microorganisms have been rapidly developed. In general, however, a protein product expressed by genetic recombination technology, in addition to the amino acid sequence designated by the gene of the protein product at the amino terminus, further contains N-formylmethioine (hereinafter referred to as 'for-Met'). Eggplants produce non-natural recombinant proteins, and E. coli is no exception. Recombinant protein does not have the same structure as the protein in the human body, so when the recombinant recombinant protein for body administration is produced by E. coli, for-Met will cause an unexpected immune response from the host, or the protein itself It was unstable and could not fully function. For example, human growth hormone composed of 191 amino acids secreted by the human pituitary gland and whose amino terminus starts with phenylalanine-proline-threonine (Phe-Pro-Thr) has been produced by genetic recombination and used in the treatment of dwarfism. However, in the recombinant human growth hormone, in addition to 191 amino acids, methionine is often left unremoved at the amino terminus, and there have been reports of unexpected antibody formation caused by human growth hormone added with methionine.

Therefore, various methods have been developed to solve the problem of the recombinant protein, for example, a method for causing methionine to be cleaved in the process of secreting the recombinant protein out of the host cell (see EP 008832, USP 4,755,464) or recombinant A method for preparing a native protein using a method of aminopeptidase, which is an enzyme that selectively cleaves only methionine located at the amino terminus of a protein, has been proposed (see WO 86/01229, WO 86/204527, EP6296695, USP 5,569,598). However, this method was able to convert the recombinant protein into a naturally occurring protein relatively simply. However, in order for the aminopeptidase to act on the recombinant protein and convert it into a native protein, only the amino terminal methionine was removed and cleaved. It is necessary to stop the reaction and convert the recombinant protein into a native protein in a small amount, and the disadvantage that the methionine removal activity of the aminopeptidase is greatly influenced by the type of adjacent amino acid following methionine, The incomplete reaction of the enzyme results in the coexistence of the amino methionine-removed and non-removed proteins.The two proteins have very similar physicochemical properties. Contains the problem of being quite difficult There.

On the other hand, in order to solve this problem, a method of linking an amino acid sequence capable of conferring specificity to the amino terminus of a protein to be expressed through a cleavage site of a specific proteolytic enzyme (see WO 89/12678, EP 20209, EP321940). When such a fusion protein is cleaved with an enzyme having specificity to the mediated sequence, the target protein having an uncleaved fusion protein and a normal amino terminal generated by cleavage can be easily separated by the difference in properties of the two substances, in which case the final purified The identity of the amino terminus is further ensured. However, in order to cleave the target protein from the expressed fusion protein, an expensive enzyme must be continuously administered to the production, and this step is a production cost and the purity and yield of the final purified protein in the process of mass production of the protein. Since it is one of the most important factors affecting the conventional cutting method as described above, there is a problem in economical mass production, and there is a need in the art to develop a method for preparing an immobilized enzyme for reuse of enzyme. This has been emerging.

In particular, efforts have been made to mass-produce a target protein using a fusion protein in which the amino terminal of the target protein is linked to a glutathione-S-transferase (GST) fragment and a urokinase cleavage site. Since urokinase used to cleave a target protein is expensive, economic production of a target protein such as human growth hormone could not be expected without reuse of the enzyme. Therefore, the need to develop a method for producing immobilized urokinase has been constantly emerging.

Accordingly, the present inventors have attempted to develop a method for producing immobilized urokinase for economically mass production of human growth hormone using GST-UK-hGH which connects the amino terminal of human growth hormone to a GST fragment and a urokinase cleavage recognition site. As a result of intensive research, the present invention has been made by semi-covalently immobilizing urokinase to the adsorption resin to prepare immobilized urokinase, and it is possible to economically cleave the target protein from the fusion protein by reusing the electric enzyme. It became.

After all, the main object of the present invention is to provide a method for producing immobilized urokinase by immobilizing urokinase semi-covalently.

1 is a graph showing the degree of cross-linking and inactivation of immobilized urokinase according to the concentration of glutaraldehyde of urokinase and bovine serum albumin (BSA) adsorbed on hydroxylapatite.

Figure 2 is a graph comparing the long-term stability of the urokinase immobilized by the semi-covalent binding method with the wild type urokinase (free urokinase).

3 is a graph comparing the thermal stability of urokinase immobilized by a covalent binding method with wild type urokinase.

Figure 4 is a SDS-polyacrylamide gel electrophoresis showing that the human growth hormone fusion protein is cleaved by a semi-covalent binding method and covalent binding method immobilized urokinase.

Figure 5 is a graph measuring the degree of cleavage of the fusion protein according to the cleavage cycle of the urokinase immobilized by a semi-covalent binding method.

In the method for preparing immobilized urokinase (UK) of the present invention, after the suspension of the bead-shaped adsorption resin in a pH 7.0 to 9.0 buffer solution, the urokinase is added to the electrosorption resin and stirred to be sufficiently adsorbed. After adding serum serum albumin (BSA) to the mixture, the empty space where UK was not adsorbed was sealed with BSA, and the UK and BSA adsorbed resins were washed with a buffer solution of pH 7.0 to 9.0, followed by glutaraldehyde. ) Is added and stirred. At this time, the stirring is performed at 1 to 10 ° C, preferably 3 to 5 ° C, most preferably 4 ° C, for 10 minutes to 100 minutes, preferably for 15 minutes to 60 minutes.

Hereinafter, the present invention will be described in detail.

In the present invention, an inclusion body expressed from recombinant E. coli MC1061 ΔpG2 (KFCC 10976), which is a strain transformed with a vector encoding a GST-UK-hGH fusion protein designed to be cleaved by urokinase. Fusion protein cleavage was performed using GST-UK-hGH fusion protein and urokinase origin. After adsorption of urokinase to hydroxylapatite (hereinafter referred to as 'HTP') resin, the activity of the immobilized urokinase prepared by chemically crosslinking was sufficiently maintained and the stability was greatly increased, and the immobilized urokinase was a batch process ( It can be reused more than 10 times by applying to batch type process).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of HTP-immobilized Eurokinase

Bead-shaped HTP (Macro-Prep hydroxylapatite II, Biorad, USA) was suspended in 20 mM Tris-HCl at pH 7.8, then 0.1 mg of urokinase (UK) per 1 ml volume of HTP was added to ensure sufficient adsorption. After stirring for 1 hour in the mixture, bovine serum albumin (BSA) was added to the mixture, and the mixture was sealed at 4 ° C. for 1 hour while blocking the empty space where UK was not adsorbed with BSA, and HTP adsorbed with UK and BSA was removed. After washing three times with 20mM Tris.HCl solution at pH 7.8, glutaraldehyde was added in concentrations ranging from 0 to 20 μl / ml and stirred at 4 ° C. for 15 minutes to ensure the integrity of UK and BSA adsorbed on HTP. Immobilized UK was prepared by increasing the integrity. Glutaraldehyde that remained unimmobilized was removed by washing with 20 mM Tris-HCl buffer at pH 7.8, and the degree of cross-linking by glutaraldehyde was 0.5 M phosphate buffer solution without reacting glutaraldehyde. Based on the UK and BSA eluted when (pH 7.0) was added, the amount of UK and BSA that was not eluted by adding 0.5M phosphate buffer solution (pH 7.0) after reacting glutaraldehyde was determined. Bradford) was calculated by the quantitative method, it is compared and expressed as a percentage (see Fig. 1). In Fig. 1, (-○-) indicates inactivity, and (-―-) indicates the degree of crosslinking. As shown in Figure 1, in the range of glutaraldehyde concentration of 3 to 10μl / ml UK showed more than 80% cross-linking efficiency with HTP, the result of measuring the specific activity of the immobilized UK in the same manner as in Example 3 to be described later , Compared with wild type UK, showed over 80% inactivity.

Example 2: Preparation of Activated CH Sepharose 4B Immobilized Eurokinase

Activated CH Sepharose 4B (Pharmacia Biotech, Sweden) was washed with 1 mM HCl and then equilibrated with buffer (pH 8.0) containing 50 mM NaHCO 3, 0.5M NaCl. After dialysis of the UK with the same buffer solution, 1 mg of UK per 1 ml of activated CH Sepharose 4B was mixed and bound at 4 ° C. for 2 hours, and the ligand remained on the surface without participating in the reaction. Ligand was removed by reaction with the addition of 0.2M glycine.

Example 3: Eurokinase Inactivity Measurement

To measure the specific activity of immobilized UK, p-Gly-Gly-Arg-p-nitroanilide (S2444, Sigma, USA) was diluted in 20 mM Tris.HCl buffer at pH 7.8 and 0.6 mM S2444 and immobilized UK were 1: After mixing at a rate of 1 and reacting with stirring at 37 ° C. for 45 minutes, the absorbance of the supernatant was measured at 405 nm. UK as a control was measured in the same manner as immobilized urokinase after dilution to a concentration of 10 to 1000 IU / ml.

Example 4 Determination of Stability of HTP-Immobilized Eurokinase

Example 1 was immobilized at a concentration of 0.1mg UK / ml HTP and wild type UK of 10μg / ml concentration aseptically stored in 20mM Tris-HCl buffer pH 7.8 at 37 ℃, taking a certain amount over time UK specific activity was measured in the same manner as in Example 3 (see FIG. 2). In FIG. 2, (-)-indicates the inactivation of wild type UK (free UK), and (--) indicates the inactivation of HTP immobilized UK. As shown in the graph of FIG. 2 comparing the long-term stability of the urokinase immobilized by the semi-covalent binding method with the wild type UK, it was confirmed that the HTP-immobilized urokinase was superior to the wild-type urokinase.

Example 5 Stability Measurement of Activated CH Sepharose 4B Immobilized UK

Activated CH Sepharose 4B immobilized UK and wild-type UK were suspended in 20 mM Tris-HCl buffer solution at pH 7.8 to 10 μg / ml, respectively, and then the activity was measured in the same manner as in Example 3 while heating at 70 ° C. See Figure 3). In FIG. 3, (-○-) represents the inactivation of activated CH Sepharose 4B immobilized UK, and (-)-represents the inactivation of wild type UK. As shown in the graph of FIG. 3 comparing the thermal stability of urokinase immobilized by covalent binding method with that of wild type urokinase, the UK immobilized on activated CH Sepharose 4B shows that the thermal stability is much higher than that of wild type UK. Could.

Example 6: Fusion Protein Cleavage Using Immobilized Eurokinase

GST in the form of inclusion bodies expressed from recombinant E. coli MC1061 ΔpG2 (KFCC 10976), a strain transformed with a vector encoding a GST-UK-hGH fusion protein designed to be cleaved by urokinase. -UK-hGH fusion protein was prepared by dissolving in 6-8 M urea and then desalting with Sephadex G-25 (Pharmacia Biotech, USA). HPT immobilized UK and wild type UK were mixed with the fusion protein and reacted at 37 ° C. for 24 hours so that the ratio of fusion protein to UK was 500: 1 (v / v). For HTP immobilized UK, each time immobilized UK was washed with 20 mM Tris.HCl buffer at pH 7.8 and fusion protein was added and the UK immobilized on activated CH Sepharose 4B was placed on a column (1 × 2.5 cm). After filling, a packed bed reactor was prepared and used repeatedly by passing a fusion protein. Proteins cleaved by immobilized UK and wild type UK were subjected to SDS-polyacrylamide gel electrophoresis (see FIG. 4), and the degree of cleavage was measured by integrating the thickness and width of each band with a Biomed program. It is expressed as a percentage based on the initial cutting (see FIG. 5). In Figure 4, lane 1 shows fusion protein GST-UK-hGH cleaved by wild type UK; Lane 2 is a fusion protein cleaved by HTP immobilized UK; Lane 3 is a non-cleaved fusion protein as a control; And lane 4 is a fusion protein cleaved by activated CH Sepharose 4B immobilized UK. As shown in FIG. 4, the cleavage ability of HTP-immobilized UK and activated CH Sepharose 4B immobilized UK was almost equivalent to that of wild-type UK, and the degree of cleavage of the fusion protein according to the cleavage cycle of urokinase immobilized by a semi-covalent binding method. As shown in the graph of FIG. 5, HTP-immobilized UK showed no reduction in cutting capacity even after repeated use 10 times, and the packed bed reactor using activated CH Sepharose 4B immobilized UK was 0.5 bed volume / hour. The cleavage reaction was possible for 3 months at the rate.

As described above in detail the specific parts of the present invention, for those skilled in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described and demonstrated in detail above, the present invention provides a method for preparing immobilized urokinase in which the wild type urokinase and a fixing resin are semicovalently bound. According to the present invention, an expensive urokinase can be reused after appropriate treatment, thereby economically cleaving and separating the target protein from the fusion protein. In particular, by successfully applied to the GST fusion protein containing human growth hormone, it is possible to economically separate the human growth hormone from the human growth hormone expressed in the form of the fusion protein, the recombination of other fusion protein forms having a urokinase cleavage site It can be widely applied to protein production.

Claims (5)

Adding urokinase to a hydroxylapatite resin suspended in a buffer solution and stirring; Adding serum serum albumin (BSA) to the hydroxylapatite resin to which the urokinase adsorbed above was obtained and stirred, followed by washing with a buffer solution; And, A method for producing immobilized urokinase comprising the step of adding glutaraldehyde to a hydroxylated apatite resin adsorbed with urokinase and bovine serum albumin obtained by the foregoing and stirring, followed by washing with a buffer solution. The method of claim 1, Eurokinase is added at a ratio of 0.05 to 1.0 mg per 1 ml of hydroxyl apatite Method for producing immobilized urokinase. The method of claim 1, Stirring is carried out at 1 to 10 ℃, characterized in that performed for 10 to 100 minutes Method for producing immobilized urokinase. The method of claim 1, The buffer solution is characterized in that 20 to 50mM Tris.HCl of pH 7.0 to 9.0 Method for producing immobilized urokinase. The method of claim 1, Glutaraldehyde is characterized in that it is added to the hydroxylapatite resin at a concentration of 2 to 30μ / ml Method for producing immobilized urokinase.