KR100309700B1 - Method for refolding recombinant protein - Google Patents

Abstract

PURPOSE: A method for refolding a recombinant protein is provided, thereby effectively activating an inactivated recombinant protein. CONSTITUTION: A recombinant protein is activated by diluting, oxidizing and reducing it using an buffer solution containing strong acid salt as an adjusting solution, in which the strong acid salt is selected from the group consisting of NaClO4, Na2S2O8, NalO4, CCl3CO2Na, Na2S2O3 and NaF, preferably NaClO4; the adjusting solution is obtained by dissolving urea and subjecting it to chromatography; the buffer solution containing strong acid salt is a tris buffer solution containing strong acid salt, an oxidizing agent and a reducing agent, wherein the concentration of NaClO4 in the buffer solution is 0.3 to 1.0 M; and the oxidizing agent is oxidized glutathione and the reducing agent is reduced glutathione.

Description

Reconstitution of Recombinant Protein into Active State

Figure 1 is a SDS-PAGE of the process of purification by chromatography in the state dissolved genetically engineered hepatitis B virus X antigen expressed in bacterial cells,

2 is electrophoresed without hepatitis B virus X antigen reconstituted by the method of the present invention without SDS,

Figure 3 is a measure of the circular dichroism (CD) value of hepatitis B virus X antigen in each of the inactive state, the state reconstituted by the conventional method, and each state reconstituted by the method of the present invention,

FIG. 4 measures the activity of hepatitis B virus X antigen in the reconstructed state of FIG. 3 by chloramphenicol acetyltransferase (CAT) activity measurement method.

The present invention relates to an effective method capable of reconstituting inactivated forms of inactivated proteins produced in microorganisms by DNA recombination techniques. More particularly, the present invention relates to a method for reconstituting an inactivated recombinant protein produced in a microorganism by DNA recombination technology, wherein the protein is reconstituted into an active form, characterized by a redox reaction in the presence of a strong acid salt. . Development of a method of reconstituting an inactive protein produced by a microorganism into an active state by DNA recombination technology is of great importance not only in basic research but also economically. Representative examples of such proteins include human growth hormone and tissues. And various antigens such as plasminogen activator (t-PA), interleukin-4, interleukin-6 and hepatitis B virus X antigen.

Among these proteins, X antigens are known to promote gene transcription [Jr.-Shin Twu et al., J. Virol. 63, 2857-2860 (1989)], the transcriptional activation action of hepatitis B virus X antigen (hereinafter referred to as "X antigen") is HIV-1-LTR [Seto et al., Proc. Natl. Acad. Sci. USA 85, 8286-8290 (1988)], or regulatory sites of Simian virus (SV-40) [Twu and Schloemer, J. Virol. 61, 3448-3453 (1987)], regardless of the type of animal or cell in which they exist (Edward Seto et al., Virology 173, 764-766 (1989)). This fact suggests that X antigens do not activate transcription by binding directly to DNA, but rather by binding to certain factors first and then to the promoter and enhancer moieties to activate transcription. Thus, a substance that inhibits the activity of hepatitis B virus X antigen will have an effect as a hepatitis B therapeutic agent.

In order to develop an activity inhibitory substance of X antigen, the development of an active X antigen is required. At Stanford Medical University, E. coli was transformed using a vector containing X antigen, followed by culturing, obtaining an inclusion body, and then using an acetic acid to reconstruct the inclusion antigen X antigen. The activity was still low and did not show the formation of secondary or tertiary structure of the protein (Jane Y. Wu et al., Cell 63, 687-695 (1990)).

Common methods of protein reconstitution include:

Mainly used methods include diluting X antigen extracted using guanidine hydrochloride or urea in a buffer solution containing an oxidizing agent and a reducing agent [Paolo Sarmientos et al., Bio / Technology 7, 495-500 (1991); Lin-Fa Wang et al., Gene 84, 127-133 (1988)], but the yield is very low to apply this method to antigen X as it is. Another method is to reconstruct the target protein in a solution containing a chaperin protein such as GroEL / S, Dnak [Vincent P. Pigiet & Barbara J. Schuster, Proc. Natl. Acad. Sci. USA 83, 7643-7647 (1986); Dorota Skowyra et al., Cell 62, 939-944 (1990); Jose A. Mendoza et al., J. Biol. Chem. 266, 13044-13049 (1991)], wherein the amount of help protein used should be at least 10 times greater than the amount of protein to be obtained, and the effect is not obtained for all proteins. In addition, a method of enhancing the activity by using a salt such as potassium chloride was used (Kathy M. Perry et al., Protein Science 1, 796-860 (1991)), but the effect on X antigen was not significant.

The main reason why these conventional reconstitution methods are not well applied to X antigens is that they contain a large amount of cysteine in the X antigen, which causes these cysteines to be oxidized in the air and to form disulfide bonds at other sites. This is because it continuously causes degeneration.

Thus, the present inventors have studied to solve the above problems, it can be seen that if the redox reaction in the presence of a strong acid salt, foreign proteins such as X antigens produced in microorganisms can be reconstituted in an active state.

On the other hand, Yuji Goto et al. Demonstrated that there is a partially active state called A state by using strong acid and salts in preactivated protein. At this time, hydrochloric acid, sulfuric acid and nitric acid are maintained at acidic pH to maintain intermediate state Acids and salts such as K ₃ Fe (CN) ₅ , Na ₂ SO ₄ , NaClO ₄ , NaCl, KCl, etc. were used, and the concentration of salt used was less than 50 mM [Yuji Goto et al., Biochem. 29, 3480-3488 (1990)], this method was used only for the demonstration of an intermediate condition and was not related to protein reconstitution. Moreover, the protein cannot be reconstituted with an active protein because the protein cannot be oxidized at acidic pH.

Accordingly, an object of the present invention is to provide a method for reconstructing a recombinant protein expressed by genetic engineering into an active form.

In the detailed description and examples of the present invention, an example of a recombinant protein is described using X antigen, but the present invention is not limited thereto. For those skilled in the art, the present invention may include DNA recombination technology in addition to X antigen. It is also applied to various kinds of foreign proteins produced by microorganisms, such as human growth hormone, tissue plasminogen activator (t-PA), interleukin-4, interleukin-6, and X antigen of hepatitis B virus. It is a well known fact.

Hereinafter, the present invention will be described in detail.

The present invention is a method of obtaining an X antigen in an active state by reconstitution characterized in that the redox reaction is performed in a buffer solution containing a strong acid after controlling the recombinant foreign protein expressed in a microorganism by a genetic engineering method.

Examples of the strong acid salt include NaClO ₄ , Na ₂ S ₂ O ₈ , NaIO ₄ , NaNO ₃ , CCl ₃ CO ₂ Na, Na ₂ S ₂ O ₃ , NaF, and the like, of which NaCl 0 ₄ is most preferred.

The modifier may be carried out using a general method such as dissolution with urea or guanidine salts, chromatography, dialysis, and the like. In an embodiment of the present invention, a process of obtaining a modifier material by continuously performing dissolution and chromatography with urea is performed. Shows.

As the buffer solution containing the strong acid salt, the buffer solution containing the strong acid salt, the oxidizing agent, and the reducing agent may be used. As the buffer, Tris is preferable. The concentration of the strong acid salt may vary depending on the type of salt. For NaClO ₄ , 0.3M-1.0M is preferred. The oxidizing agent and the reducing agent may be oxidized dithiothreitol (DTT) and reduced DTT, oxidized glutathione and reduced glutathione, and the concentration of the reducing agent is 2-5 times higher than the oxidizing agent, but the concentration of the oxidizing agent It is preferable that the farmhouse of 0.5-2 mM and a reducing agent is 1-5 mM. The pH of the buffer solution is preferably weakly basic, more preferably pH 8-9. In order to prevent precipitation of the protein during the redox reaction, it is preferable to include urea in a buffer solution, and the concentration is suitably 1-3M.

After diluting the crude solution with a buffer solution, a redox reaction is performed. The dilution temperature does not significantly affect the dilution, but it is more preferable to dilute the crude liquid in an ice bath than when diluting at room temperature. Dilution increases efficiency by diluting to a final concentration of 100 µg / ml or less, more preferably 50 µg / ml or less. The temperature of the redox reaction is preferably 20-25 ° C, and the reaction time may be at least 2 hours or more, but preferably 10-40 hours.

The reaction solution was concentrated to about 10-20 μM and dialyzed with a buffer solution without urea to obtain X antigen having a physiological activity.

The protein thus obtained can be quantified to determine the yield of the final protein obtained from the crude liquid. Protein quantitation is described, for example, by the Bradford method [Bradford, M. M., Anal. Biochem. 72, 248-254 (1976).

When the recombinant protein is reconstructed by the conventional method without using the strong acid salt, the presence of the strong acid salt is absolutely necessary because most of the protein precipitates during this dialysis process.

Finally, the secondary structure of the reconstituted recombinant protein can be confirmed by measuring ion exchange resin, electrophoresis in unmodified state, and CD value.

In addition, in order to know whether the protein reconstituted by the present invention is correctly reconstituted in the active state, various methods can be used according to the activity of the protein, for example, in the case of X antigen can be known by measuring the DNA transcription activity have. DNA transcription activity measurement is performed by directly processing samples to be measured in a culture medium of an animal cell into which a vector containing a CAT gene, such as pSV2CAT (purchased from Fox Chase Cancer Center), is measured, and then measuring the activity of the resulting CAT enzyme. Is done.

pSV2CAT has the E promoter of SV40 virus, which is one of the DNA structures required for hepatitis B virus X antigen to be active, at the transcriptional regulatory region of the CAT (chloramphenicol acetyl transferase) gene. When present the expression of CAT enzyme will increase. CAT enzyme activity was measured using chloramphenicol conjugated with radioisotope ¹⁴ C [Gorman, CM & Howard. BH, Mol. Cell. Biol. 2, 1044-1051 (1982).

Hereinafter, the present invention will be described in more detail with reference to Examples.

The following examples are merely illustrative of the present invention and the present invention is not limited by the following examples.

REFERENCE EXAMPLE 1 Culture of Bacterial Cells Producing Hepatitis B Virus X Antigen

Recombinant hepatitis B virus X antigen producing strain E. coli BL21 (DE3) pET8c-HBX pLYS. S (obtained from Stanford Medical School Professor William S. Ronbinson) was shaken in 2 ml of Luria medium with ampicillin and chloramphenicol for 8 hours, and then transferred to 100 ml Luria medium with ampicillin and chloramphenicol for 12 hours. Shake culture was used as seed culture (seed culture) for fermentation.

The fermentation was carried out in a Luria medium containing 50 μg / ml of ampicillin and 25 μg / ml of chloramphenicol at a shaking speed of 500 rpm, dissolved oxygen amount of 1 vvm, incubation temperature of 37 ° C., and pH 7.0 using a 10 L-bench tower fermenter.

After 3-4 hours of fermentation, the promoter of the recombinant hepatitis B virus X antigen expression vector was added by adding 0.4 mM IPTG (isopropyl-β-D-thiocalactopyranoside) to the medium at 0.5 nm of absorbance at 600 nm. To work.

Three hours after the addition of IPTG, the cell culture was centrifuged at 5000 rpm for about 10 minutes with a centrifuge (Beckman Rotor JA-10), and then bacterial cell precipitates were frozen at -70 ° C.

Reference Example 2 Adjustment Process

30 g of bacterial cells containing the hepatitis B virus X antigen obtained in Reference Example 1 were suspended in 150 ml of a buffer solution (50 mM Tris-Hcl, pH 7.5, 5 mM EDTA, 100 µg DNase I) to destroy bacteria. This is based on the properties of the pLYS.S vector. The lysed protein was then removed by centrifugation at about 30,000 g for 30 minutes using a centrifuge. The precipitate was washed with a washing solution (0.2 mg / ml lysozyme, 1 mM EDTA, 1 mg / ml deoxycholate), and then centrifuged at 30,000 g for 30 minutes to obtain a precipitate. After the above washing process was repeated two more times, the precipitate was stirred for 30 minutes at 150 rpm at 4 ° C. with 150 ml of a dissolving buffer solution (30 mM phosphate, pH 6.5, 8M urea, 20 mM DTT). The suspension thus obtained was centrifuged at 40,000 g for 30 minutes to remove insoluble precipitate.

The supernatant was dialyzed with chromatography buffer (30 mM phosphate, pH 6.5, 8 M urea, 1 mM DTT), and then the sample was absorbed into a CM-Sepharose column (5 cm x 15 cm) equilibrated with the same buffer and 0.1-0.3 M NaCl Eluted with a linear concentration gradient of. The fragments obtained at this time were collected by SDS-PAGE to collect fragments with X antigen, which were then concentrated to 10 mg / ml using a YM3 membrane (Amicon).

The concentrated sample was subjected to gel filtration chromatography using a Sephaac S-200 column equilibrated with a buffer solution (30 mM phosphate, pH 6.5, 8M urea, 1 mM DTT), and then each fragment was subjected to SDS-PAGE and at least 95%. Fragments with purity were collected. It was concentrated to 2 mg / ml using YM3 membrane (Amicon) and used as a sample for the next reconstitution.

SDS-PAGE of each sample of the above adjustment process is shown in FIG.

The first column of FIG. 1 is standard proteins of size 18, 27, 32, 49, 80, 106 KD, the second column is the inclusion body dissolved in urea, and the third column collects fragments after the CM-Sepharose column. The fourth column is the fragments collected after the Sephacryl S-200 column.

EXAMPLES Reconstitution with NaCl0 ₄

The adjusted hepatitis B virus X antigen was diluted in a 40-fold volume buffer (20 mM Tris-HCl, ph 8.5, 1.8 M urea, 0.5 M NaClO ₄ , 1 mM oxidized DTT, 2 mM reduced DTT) in an ice bath. After leaving for 30 minutes, it was transferred to 22 ° C. and left for another 15 hours.

The diluted and left samples were concentrated to 1/10 volume with YM10 membrane (Amicon), and then the buffer solution (20mM Tris-HC1, pH 8.5, 0.5) containing 1.5, 1.0, 0.5, and 0 M of urea, respectively. Urea was removed by dialysis sequentially at 4 ° C. with M NaClO ₄ , 1 mM oxidized DTT, 2 mM reduced DTT).

Finally, dialyzed at 4 ° C. with oxidized DTT-free buffer (20 mM Tris-HC1, pH 8.5, 0.5 M NaClO ₄ , 1 mM reduced DTT), the precipitate was removed with a 0.22 μm filter (Millipore), and then the protein The yield was determined by quantification.

The yield of reconstitution by the above method was about 25-30%.

Comparative Example 1 Reconstitution without Salt

The adjusted hepatitis B virus X antigen was diluted in a 40-fold volume buffer (20 mM Tris-HCl, pH 8.5, 1.8 M urea, 1 mM oxidized DTT, 2 mM reduced DTT) in an ice bath and left for 30 minutes. It was transferred to 22 ° C. and left for another 15 hours.

The diluted and left samples were concentrated to 1/10 volume with YM10 membrane (Amicon), and then the buffer solution (20mM Tris-HCl, pH 8.5, 1mM) containing 1.5, 1.0, 0.5, and 0 M of urea, respectively. Urea was removed sequentially by dialysis at 4 ° C. with oxidized DTT, 2 mM reduced DTT). When X antigen is reconstituted by the above method, most of the precipitate is precipitated in the dialysis process, and the yield by the above method is 1% or less.

Comparative Example 2 Reconstitution Using Potassium Chloride

The adjusted hepatitis B virus X antigen was diluted in a 40-fold volume buffer (20 mM Tris-HCl, pH 8.5, 1.8 M urea, 0.5 M KCl, 1 mM oxidized DTT, 2 mM reduced DTT) in an ice bath, 30 After being left for a minute, it is transferred to 22 ° C. and left for another 15 hours. The diluted and left samples were concentrated to 1/10 volume with YM10 membrane (Amicon), and then the buffer solution (20 mM Tris-HC1, pH 8.5, 0.5) containing urea 1.5, 1.0, 0.5, and 0 M, respectively. Urea was removed sequentially by dialysis at 4 ° C. with M KC1, 1 mM oxidized DTT, 2 mM reduced DTT). This dialysis produced a greater amount of precipitation, though less than without salt.

Finally, dialyzed at 4 ° C. with oxidized DTT-free buffer (20 mM Tris-HCl, pH 8.5, 0.5 M NaCl0 ₄ , 1 mM reduced DTT), the precipitate was removed with a 0.22 μm filter (Millipore) and the protein The yield was determined by quantification. The yield by the said method was about 10%.

Test Example 1 Confirmation of Secondary Structure of Reconstituted Hepatitis B Virus X Antigen

A portion of the reconstituted sample was dialyzed at 4 ° C. with NaC10 ₄ -free buffer (20 mM Tris-HC1, pH 8.5, 1 M urea, 1 mM reduced DTT) and equilibrated with the same buffer solution (DE-52, Whatman), and eluted with 0.5M NaClO ₄ . Unlike the case where the X antigen modified by 8M urea is adsorbed on the cation exchange resin, most of the X antigens are adsorbed on the anion exchange resin in this case. This is a reconstitution method according to the present invention, compared with the adsorption of an X antigen having an activity obtained from E. coli to an anion exchange resin using a vector having an X antigen gene linked to a maltose binding protein (MBP) gene. It can be seen that this does not merely increase the protein yield.

SDS-free gels were prepared using acetic acid-KOH (pH 4.3) buffer, and the reconstituted X antigen obtained in the above example was electrophoresed using electrophoretic buffer (acetic acid-β-aniline, pH 4.5). It was run (figure 2). Unlike the shape of the sample reconstituted by the previous method without electrophoresis without SDS, the sample of the reconstituted X antigen showed a protein band that is expected to be homogeneous.

The secondary structure of the reconstituted X antigen obtained in the above example was confirmed by measuring the CD value of the sample using a J-600 Spectropolarimeter (JASCO, Japan) instrument. X antigen reconstituted with NaC10 ₄ (0.5M) in Tris-HCl (pH 8.0, 2mM) and X antigen reconstituted in Tris-HCl (pH 8.0, 2mM) by conventional methods, and modified with 8M element CD values of X antigens were measured at the same concentration (13.3 μM) (FIG. 3). The upper first line of FIG. 3 is inactivated by urea, the center line is reconstructed by the conventional method without using salt, and the bottom line is the CD value of the X antigen in the reconstructed state by the method of the present invention. Each is measured.

The CD value of the X antigen reconstituted with NaC10 ₄ at 220 nm shows the largest negative value, and the reconstitution is based on the secondary structure values of five proteins (myoglobin, lysozyme, ribonuclease A, papain, cytochrome C). An X antigen was found to have a 36.4% α structure and a 37.7% β structure.

Test Example 2 Physiological Activity Measurement

CV-1 (ATCC CCL 70) cells, an African green monkey male kidney cell, were cultured in a 6 cm diameter culture vessel containing 5 ml of DMEM medium (Dulbecos Modification of Eagle's Medium; DIFCO # 12800-066), and the cells were cultured. When grown to about 70-80% of the surface, a vector containing the CAT gene (pSV2CAT) was injected.

Vector injection was performed using DOTAP (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammoniummethylsulfate, Boehringer Mannheim). Dilute 30 μl DOTAP with 70 μl dilution buffer HBS (Hepes, 20 mM; NaC1, 150 mM; pH 7.4), mix it slowly with 100 μl of pSV2CAT (5 μg) diluted with the same solution, and then at room temperature for 15 minutes. It was left to. 5 ml of DMEM medium was added to the solution, which was placed in a culture vessel washed with PBS, and then incubated at 5-7% CO ₂ , 37 ° C. for 20 hours.

After the vector-injected animal cell culture was washed with buffer PBS (pH 7.2), a medium containing chloroquine was added to the culture medium to a final concentration of 10 μM, and 2-3 μg of the X antigen protein to measure activity was directly added. Was treated. Five hours after X antigen treatment, the medium was changed to fresh medium and incubated for another 40 hours, after which cells were collected and CAT enzyme activity was measured (FIG. 4). In FIG. 4, CM is chloramphenicol, 1,3-CM is chloramphenicol having two acetyl groups bonded thereto, the first column is unprocessed vector and protein, the second column is processed only pSV2CAT vector, and the third column. Is a combination of the pSV2CAT vector and the reconstituted protein.

Claims (15)

A method of reconstituting an inactivated foreign protein produced in a microorganism by an active DNA recombination technique into an active form, the method comprising diluting, oxidizing and reducing a crude solution using a buffer solution containing a strong acid salt. . The method of claim 1, wherein the strong acid salt is selected from NaClO ₄ , Na ₂ S ₂ O ₈ , NaIO ₄ , CCl ₃ CO ₂ Na, Na ₂ S ₂ 0 _3, and NaF. The method of claim 2, wherein the strong acid salt is NaClO ₄ . The method according to claim 1, wherein the crude liquid solution is obtained by performing a dissolution and chromatography step using urea continuously. The method of claim 1, wherein the strong acid-containing buffer solution is characterized in that the tris buffer solution containing a strong acid, an oxidizing agent and a reducing agent. 6. The method of claim 5, NaClO ₄ concentration of the strong acid salt is NaClO ₄ wherein the buffer solution is characterized in that the 0.3M to 1.0M. 6. The method of claim 5, wherein oxidized and reduced agents are oxidized dithiothreitol and reduced dithiothreitol, or oxidized glutathione and reduced glutathione, respectively. 6. The method of claim 5 wherein the concentration of oxidant is 0.5-2 mM. The method of claim 5 wherein the concentration of the reducing agent is 1-5 mM. The method of claim 1, wherein the pH of the buffer solution is characterized in that 8 to 9. The method of claim 1 wherein the buffer comprises urea. The method of claim 1, wherein the final concentration of the foreign protein at dilution is 100 μg / ml or less. The method of claim 1 wherein the temperature at dilution is 0-4 ° C. The method of claim 1, wherein the temperature of the oxidation and reduction reaction is 20-25 ° C. The method of claim 1, wherein the oxidation and reduction time is 2 to 40 hours.