KR20020072349A - Method of Reactivating Immobilized Urokinase Column - Google Patents

Abstract

PURPOSE: A method for reactivating an immobilized urokinase column is provided, thereby recycling immobilized urokinase column to obtain a target protein and consequently reducing costs therefor. CONSTITUTION: The method for reactivating a urokinase immobilized column comprises the steps of: passing a fusion protein through a urokinase covalently bound column to cut the fusion protein; inserting a urokinase specific mutagen into the fusion protein passed through the urokinase immobilized column to unfold a protein structure; and removing the urokinase specific mutagen from the column to refold the urokinase, in which the mutagen is guanidine HCl, urea and ion type surfactant; the column is filled with a resin consisting of sepharose gel; and the mutagen is removed by inserting a buffer into the column.

Description

Regeneration of Immobilized Eurokinase Columns {Method of Reactivating Immobilized Urokinase Column}

The present invention relates to a method for regenerating an immobilized urokinase column, and more particularly, to an immobilized urokinase column for repeatedly using an immobilized urokinase column for cutting and isolating a fusion protein containing a target protein to obtain a target protein. It relates to a regeneration method.

With the development of genetic recombination technology, a method for mass production of various useful proteins, particularly biomedical proteins, has been developed using microbial expression systems. Recently, studies have been conducted on a method of expressing a target protein in the form of a fusion protein to increase expression efficiency by a host cell or improving stability and purification characteristics, and cutting the expressed fusion protein to obtain a target protein. Actively in progress The process of cleaving and separating the target protein from the expressed fusion protein using proteolytic enzymes is one of the important effects on the production cost of the target protein production process and the purity and yield of the final purified protein.

A commonly known method for cleaving fusion proteins is a batch reaction in which proteolytic enzymes and fusion proteins are introduced into a cleavage reactor to cleave the fusion protein. However, when the cleavage reaction is carried out in a batch manner as described above, it is difficult to recover and reuse the enzyme because the reactants, products, and enzymes are mixed in the reactor after the enzymatic reaction. To offset the advantages of In addition, it is easy to inactivate enzymes due to shear stress caused by stirring, and there is a risk of causing enzyme inhibition by the product produced after the enzyme reaction. impossible.

Therefore, if the fusion protein is cleaved and separated using an immobilized enzyme reaction to immobilize the enzyme on a solid phase, the enzyme can be easily reused, thereby greatly reducing the cost of the enzyme, and also facilitating recovery and purification of the target protein after the cleavage reaction. The economics of the protein cleavage process can be greatly improved. In addition, the immobilized enzyme has improved stability to pH and temperature, and has the advantage of having high productivity through a high concentration enzyme reaction and a continuous reaction. However, after the enzyme is immobilized on the solid phase to be filled in a column, and the cleavage and separation of the fusion protein are performed, impurities or enzyme products such as cell debris in the fusion protein bind to the enzyme immobilized on the solid phase to inhibit the enzyme ( There is a problem that inhibits the repeated use of the immobilized enzyme, because it causes the action.

Therefore, the cleavage method of the fusion protein using proteolytic enzymes is not widely used in large-scale processes because of the high cost required for purification of the target protein and proteolytic enzymes despite the above-mentioned advantages.

It is an object of the present invention to provide a method for regenerating an immobilized urokinase column which can economically and simply perform a cleavage process of a fusion protein.

It is another object of the present invention to provide a method for regenerating an immobilized urokinase column which can enlarge the cleavage process of a fusion protein on an industrial scale.

Still another object of the present invention is to provide a method for regenerating an immobilized urokinase column for repeatedly using an immobilized urokinase column for cleaving and isolating a fusion protein comprising a target protein to obtain a target protein.

1 is a graph showing the activity of urokinase during one cycle of supplying denaturant and buffer to an immobilized urokinase column.

Figure 2 is a graph showing the activity of urokinase during the 22 times the process of supplying denaturant and buffer to the immobilized urokinase column.

Figure 3 is a photograph showing the SDS-PAGE of each step solution to purify the hGH monomer.

In order to achieve the above object, the present invention is a process of cleaving the fusion protein by passing the fusion protein through a solid column covalently bonded to the urokinase, injecting a urokinase specific denaturant into the immobilized urokinase column passed through the fusion protein. The present invention provides a method for regenerating an immobilized urokinase column, which includes solving a protein structure of urokinase, and regenerating urokinase by removing a denaturant from the column. Here, the denaturant is selected from the group consisting of ionic surfactants such as guanidine HCl, urea, sodium dodecyl sulfate (SDS), and the fusion protein is selected from human growth hormone. It is preferred if the glutathione S transferase is fused by a linker comprising a urokinase cleavage site.

Hereinafter, the present invention will be described in detail.

Serine protease-based urokinase (UK), which is commonly used as a thrombolytic agent, has been used as a cleavage enzyme such as enterokinase, thrombin, and factor Xa. mass production from urine) or mass production using a prokaryotic expression system such as Escherichia coli.

In the present invention, a urokinase capable of separating and cleaving a fusion protein is used by attaching it to a solid-phase column. As such, the solid phase column to which urokinase is attached is prepared by filling urokinase in a column including a resin whose surface is activated such that urokinase is covalently bonded. For example, in the case of using Sepharose gel as a resin, the cepharose gel is etherified using glycidol and etherified with sodium periodate (NaIO ₄ ), an oxidizing agent. After oxidizing the gel and activating it, the immobilized UK column is prepared by covalently binding the aldehyde group formed on the surface of the gel with the amine group of the urokinase. Activated resins to which urokinase can be bound include Sepharose gels such as Sepharose 4B, Sepharose 6B, Sepharose CL-4B, Sepharose CL-6B (manufactured by Amersham Pharmacia Biotech, Uppsala, Sweden) and the like. You can use the one activated by the method.

When the cell lysate containing the fusion protein is passed through the prepared urokinase-coated solid-phase column, the fusion protein is separated and cleaved by the enzymatic action of the urokinase, and is discharged from the column. In this process, proteins separated from impurities such as cell debris do not completely flow out of the solid phase column, but remain in the column in combination with urokinase. Thus, impurities such as cell debris inhibit the activity of urokinase. do.

As such, when the urokinase specific denaturant is added to the immobilized urokinase column whose activity is reduced through cleavage of the fusion protein, the protein structure of urokinase is unfolded. In this case, various denaturing agents that can solve the protein structure of urokinase can be used, but ionic types such as guanidine HCl, urea, and sodium dodecyl sulfate (SDS) can be used. Use of such as the surfactant is preferable because it can effectively solve the protein structure of urokinase. Here, the concentration of the denaturant may be appropriately selected depending on the type and density of the column, but in general, in the case of guanidine HCl or urea, it is preferable to use an aqueous solution of 4 to 10 M, and an ion such as sodium dodecyl sulfate (SDS). In the case of a type surfactant, it is preferable that it is a 10-50mM aqueous solution, and it is preferable that the input time of the said modifier is 30 minutes-1 hour. While the protein structure of urokinase is released by the denaturant, cell debris or cleaved protein that aggregates with urokinase is separated from urokinase and eluted.

When the denatured agent is used to solve the protein structure of the urokinase, and then the buffer is added to remove the denatured agent from the column, the urokinase is refolded and the remaining cell debris or cleaved protein is separated from the urokinase to completely remove from the column. The immobilized Eurokinase column can be regenerated. In this case, a buffer generally used (for example, 0.1 M phosphate buffer) may be widely used, and the input time of the buffer for removing the denaturant is preferably 1 to 3 hours.

As such, the desired protein can be obtained by separating and purifying the protein cleaved and separated by the immobilized urokinase by a conventional process such as centrifugation and expansion layer adsorption chromatography, and can be cleaved and separated by the immobilized urokinase according to the present invention. One possible fusion protein is a fragment of GST (glutathione S transferase) at the C-terminus of a human growth hormone fused by a linker (linker) comprising a urokinase cleavage site. Fused) can be used.

Next, examples are provided to help understanding of the present invention. However, the present invention is not limited by the following examples.

EXAMPLE

The urokinase used in the examples (hereinafter referred to as UK, provided by Green Cross Co., Ltd.) has an activity of 352,000 IU / ml, and the fusion protein of GST (glutathione S transferase) at the C-terminus of human growth hormone is Fragments (69 amino acids) were fused with a linker having the amino acid sequence of Gly-Thr-Gly-Arg as a UK cleavage site (Cooke, NE, et al. J. Biol. Chem. , 263) . (18), 9001-9006). Sepharose CL-6B was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden) and used as a gel activator glycidol (2,3-epoxy-1-propanol) (glycidol (2,3-epoxy) -1-propanol), sodium periodate and sodium borohydride were all purchased from Sigma (St. Louis, USA). Reagents such as N-α-acetyl-L-lysine methyl ester hydrochloride (ALMe), perchloric acid and chromotropic acid, which are substrates for measuring UK activity, were also purchased from Sigma. .

Measurement of the activity of liquid and immobilized UK was carried out using an esterolytic method (see Barlow, GH Methods in Enzymology (1976), 45, 239) after taking a small sample from the column after the immobilization and cleavage reactions. -244) and the UK titer is expressed in CTA units (1 IU of the UK is defined as degrading 5 × 10 ⁻⁴ μM ALMe for 1 min at 37 ° C.).

(end). Activation of Sepharose Gel

Sepharose 6B-CL gel was etherified with glycidol and oxidized with sodium periodate (NaIO ₄ ), an oxidizing agent, to form an aldehyde group on the surface of the gel. Immobilized UK columns were prepared by covalently bonding aldehyde groups on the gel surface (Suh, CW et al. Korean J. Biotechnol. Bioeng. (2000), 15 (1), 42-48; Guisan JM, Enzyme Microb.Technol (1988) ), 10, 375-382; Guisan JM et al., Biotech Bioeng. (1991), 38. 1144-1152; Blanco, RM et al., Enzyme Microb.Technol. (1988), 10, 227-232; Blanco , RM et al., Enzyme Microb.Technol. (1989), 11, 353-359; Shainoff, JR, Biochem, Biophys.Res . Comm. (1980), 95 (2), 690-695).

(I). Repeated use of unfixed and refolded stationary phases in the UK

6 M guanidine HCl (Gu HCl) as a UK denaturant was supplied to a solid UK column for 30 minutes, and the denaturant was washed with 0.1 M phosphate buffer for 1 hour while taking a portion of the resin from the column to change UK activity. Was measured, and the results are shown in FIG. As shown in FIG. 1, almost all of the UK activity was lost by the injection of GuHCl, but the UK activity was restored to 98% of the initial level as a result of washing the GuHCl for 1 hour to induce refolding. From the above results, it can be seen that the yield of refolding in the immobilized state is much better than refolding in solution phase, and this phenomenon is refolded in the same fusion protein attached to the solid carrier by anion bonding. This is consistent with the results obtained by performing much better yield than solution phase refolding. Given that the number of disulfide bonds in the UK is 12, the number of disulfide bonds in the protein is not the only factor determining the refolding yield, and the primary structure of the protein is considered to be a more important factor, and also 12 disulfides The high stability of the UK following binding is also believed to contribute to the higher refolding yield. Since no UK titer was detected in the column eluate, it can be seen that the covalent bond between the UK and agarose is maintained despite the GuHCl treatment.

Since the economics of the immobilized enzyme column depends on the number of times the column can be used repeatedly, the titer of the UK was measured by repeatedly performing UK solid phase unfolding-refolding using GuHCl, and the results are shown in FIG. 2. . In each unfolding-refolding process, 6M GuHCl was introduced into the column for 1 hour to release the UK, and the re-folding was induced by washing the denaturant for 2 hours with 0.1M phosphate buffer. It can be seen from FIG. 2 that the UK titer maintains about 80% of the initial titer even when the column is repeated up to 22 times. This significantly improves the lifespan and process economics of immobilized UK columns and could be used for cleavage reactions with other expensive cleavage enzymes.

(C) Fusion Protein Cleavage and Isolation of Monomers by Immobilized UK Columns

The fusion protein was introduced into the prepared immobilized UK column to perform cleavage reaction of the fusion protein, and then the unreacted fusion protein, the hGH monomer as a target protein, and the GST fragment were eluted together. The acid precipitation method was used to separate only the hGH monomer which is the target protein from this mixture. When the pH of the eluate was adjusted to 3.5, the hGH monomer was separated into the supernatant and the fusion protein and the GST fragment were separated into precipitates. If the sediment is separated by centrifugation after acid precipitation, the process becomes manual and there is a risk of denatured hGH due to long exposure to an acidic environment, so expanded bed adsorption (EBA) chromatography (trade name: Streamline 25, Amersham) Pharmacia Biotech, Uppsala, Sweden) to remove the precipitate and recover hGH. The operating conditions of the EBA chromatography column filled with Sepharose SP-XL resin were 0.1M sodium phosphate (pH 3.5) and 10 ml / min, respectively, and the elution buffer and flow rate, respectively. 8 ml / min with 1 M NaCl. Through EBA, hGH monomer was adsorbed into the EBA column and the precipitate was eluted to separate the hGH monomer. Thus, by adjusting the eluate in the cleavage reaction by the immobilized UK column to pH 3.5 and passing through the EBA column, the hGH monomer was recovered in high yield and purity, and at the same time, the precipitated foreign matters could be eluted out of the column.

Figure 3 is a photograph showing the SDS-PAGE of each step solution for purification of hGH monomer. In FIG. 3, lane 1 is a fusion protein, lane 2 is a hGH monomer, lane 3 is a cleaved protein isolated, lane 4 is an acid-precipitate of a column eluate, lane 5 is a NaCl eluate, and lane 6 is a sample filling step. The eluate of As can be seen from FIG. 3, the hGH monomer was adsorbed to the column in high yield by cation exchange, and the adsorbed monomer was eluted by NaCl in high purity and yield (almost 100%) (lane 5). There was little loss due to flow (lane 6). That is, when the unadsorbed eluate was centrifuged after column injection, no protein was detected in the supernatant (nothing appears in the SDS-PAGE picture: see FIG. 3). Therefore, it can be seen that the target protein can be obtained in a continuous process by cleavage of the fusion protein by immobilized UK column, foreign matter precipitation by acid precipitation using pH control, precipitate removal using EBA chromatography, and monomer adsorption purification.

(D) repeated use by loosening and refolding of the immobilized UK;

6M GuHCl was injected into the immobilized UK column after the cleavage reaction of the fusion protein for 30 minutes at a flow rate of 1 BVH (bed volume per hour) to denature and elute non-exudates such as cell debris and cleaved protein accumulated in the column. The immobilized UK was converted to the loose structure. The column is then washed with 0.1 M phosphate buffer (pH 7.5), 1 BVH for 1 hour to slowly remove GuHCl to restore the three-dimensional structure of the protein in the state of immobilization on the resin. -phase refolding) 'response. Trace amounts of immobilized UK samples were taken after refolding and analyzed for titer to confirm that urokinase obtained the original three-dimensional structure.

(E) UK immobilization and cleavage yield of scaled up columns

In order to confirm the change of the fusion protein cleavage efficiency according to the volume of the immobilized UK column, the yield of the immobilization reaction and the fusion protein cleavage reaction was measured for the UK immobilization column having a volume of 6 ml and 250 ml, the results are shown in Indicated.

Column bed volume 6 ml 250 ml Concentration of Activated Aldehyde Group at Gel Surface 54 μM / ml 60 μM / ml Yield of UK Immobilization Reaction 98% 99% Yield of Fusion Protein Cleavage 50-60% 58%

From Table 1, it can be seen that even after scaling up the volume of the column to 250 ml, the UK immobilization reaction yield is almost no difference of 98% vs. 99% of the total amount of the injected UK, which indicates that the -OH group on the surface of the Sepharose gel- It is judged that the yield of the activation reaction to switch to the CHO group is maintained at about the same level regardless of the column volume (54 vs. 60 μM / ml). In addition, when the same conditions were maintained, the yield of the cleavage reaction was almost 50-60% (6 ml scale) vs. 58% (250 ml scale). This is because it is not disturbed.

(E) Economic Analysis of Immobilized UK Columns

The immobilized UK system was about 70% cost effective compared to the free UK system when the cleavage reaction was performed 20 times at 250 ml scale (UK cost was estimated at 10,000 won per 1 mg), which is a material cost. 12% of the total manufacturing cost of hGH, including labor, labor and depreciation

As described above, the method for regenerating an immobilized urokinase column according to the present invention continuously performs economical immobilized enzyme cleavage process by repeatedly performing foreign material removal and reactivation of protein activity through unwinding / refolding of urokinase. To be done. In addition, the regeneration method of the present invention not only enables to enlarge the cutting process of the fusion protein on an industrial scale, but also has an effect of extending the service life of urokinase.

Claims (6)

Cutting the fusion protein by passing the fusion protein through a solid-phase column to which urokinase is covalently bound; Injecting a urokinase specific denaturant into the immobilized urokinase column which has passed the fusion protein to solve the protein structure of urokinase; And A method for regenerating an immobilized urokinase column comprising the step of removing the denaturant from the column and refolding the urokinase. The method of claim 1, wherein the denaturant is selected from the group consisting of guanidine HCl, urea, and ionic surfactants. The method for regenerating an immobilized urokinase column according to claim 1, wherein the column is filled with a resin made of a sepharose gel. The method for regenerating an immobilized urokinase column according to claim 1, wherein the step of removing the denaturant from the column is performed by introducing a buffer into the column. 5. The method of claim 4, wherein the buffer is a phosphoric acid solution. The method of claim 1, wherein the fusion protein is a fusion of human growth hormone and glutathione S transferase.