KR100307186B1 - Purification method of ubns4e12-ii - Google Patents

Abstract

PURPOSE: Provided is a purification method of UBNS4E12-II which is a fusion protein of KHCV epitope and is manufactured from recombinant E.coli into a form of inclusion body. CONSTITUTION: The purification method of UBNS4E12-II expressed in recombinant E.coli comprises the steps of: cell lyzing the recombinant E.coli cells; removing soluble proteins; recovering and washing a protein inclusion body; dissolving the inclusion body; performing ion exchange chromatography and precipitating proteins by using and ammonium sulfate; washing the precipitates then performing gel filtration chromatography; and dialyzing them twice.

Description

Method for Purifying UBINS412-II (UBNS4E12-II), a Fusion Protein Antigen Determinant of Korean Hepatitis C Virus

Figure 1 shows an overview of the purification process for purifying UBNS4E12-II protein from recombinant E. coli according to the present invention,

Figure 2 shows the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the crude product and the final purified UBNS4E12-II protein obtained in each step of the purification process according to the present invention,

Figure 3 shows the results of confirming the eluate obtained by ion exchange chromatography of the inclusion body solution by SDS-PAGE,

Figure 4 shows the chromatogram obtained by gel filtration HPLC of purified UBNS4E12-II protein,

Figure 5 shows the results of measuring the density of purified UBNS4E12-II protein by ultrafast centrifugation using cesium chloride,

FIG. 6 shows a comparison of the CD (circular dichroism) spectrum before and after removing urea in UBNS4E12-II protein solution.

7-A shows a chromatogram of gel filtration chromatography using S-200 resin,

7-B shows the chromatogram of gel filtration chromatography using Sepharose CL-4B resin,

8 shows the result of confirming the precipitation strength of the protein by ammonium sulfate (15%) according to the pH of the solution by SDS-PAGE.

Figure 9 shows the results obtained by SDS-PAGE to determine the precipitation of different proteins in the concentration of ammonium sulfate at pH 5 · 6,

Figure 10 shows the amino acid sequence of the NA4E, E1G and E2E protein contained in the UBNS4E12-II protein and the nucleotide sequence encoding it.

The present invention relates to a method for purifying UBNS4E12-II protein, which is a recombinant specific antigen of Korean type hepatitis C virus (KHCV), produced as an inclusion body in recombinant E. coli.

Viral hepatitis is a liver disease identified by infection of hepatitis virus, the main pathogens of which are hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) and Epstein Barr virus (EBV) and the like have been identified. In early 1973, after HAV and HVB were identified and partial specificity studies were conducted, diagnostic methods for these viruses have steadily developed. However, despite the improvement of diagnostic methods, hepatitis symptoms have been consistently identified in patients who are negative for HAV and HBV, and it has been speculated that viral pathogens other than HAV and HBV exist. In 1975, the term non-A non-B Hepatitis (NANBH) was first used to describe a case of hepatitis that is not caused by HAV or HBV and related to blood transfusion (Feinstone et. al., New Engl. J. Med. 292, 454-457 (1975). Since then, research on non-AB hepatitis B virus (NANBH virus) has been focused and isolated and identified in 1989 (Choo, QL et al., Science 244, 359-362 (1989)), NANBH virus accounts for about 90% of hepatitis infected by transfusion (Alter, HJ, et al., Lancet 2, 838-841 (1975) Furthermore, it is known that 1 to 6% of donors are considered carriers of NANBH, and it is known that 40 to 50% of infected patients develop chronic hepatitis and 20% of them progress to cirrhosis and liver cancer (Dienstag, JL). and Alter, HJ, Semin.Liver. Dis. 6, 67-81 (1986)). It has been reported that plasma can be infected by chimpanzees to isolate and recover the NANBH virus (Breadley, DW et al., Gastroenterology 88, 773-779 (1985)). It is reported that it has one open reading frame (ORF) consisting of about 10,000 bases and encoding a protein consisting of about 3010 to 3011 amino acids (Choo, QL et al., Science 244, 359-362 (1989)). Choo, QL et al., Proc. Natl. Acad. Sci. USA 88, 2451-2455 (1991).

Based on these findings, the applicant fused the envelope protein antigen-determining sites of the entire gene of Korean-type HCV with the antigen-determining sites of the non-structural 4 protein in order to develop a diagnostic reagent for Korean HCV. Recombinant proteins UBNS4E1E2 and UBNS4E12-II were expressed (see Applicant's prior Korean Patent Application No. 93-7230 and Patent Application No. 94-1237, same as the present application) and have applied for a method for purifying UBNS4E1E2 protein ( See Applicant's prior Korean Patent Application No. 93-7229).

The UBNS4E12-II protein expressed above (a protein in which ubiquitin, NS4E, E1G and E2E proteins are fused in turn: FIG. 10) is expressed in E. coli in the form of an inclusion body. The inventors have begun research using possible methods that can be used to purify recombinant proteins expressed as inclusion bodies in Escherichia coli generally to develop a method for purifying expressed UBNS4E12-II proteins. To this end, after inclusion body dissolution, anion exchange chromatography and cation exchange chromatography were performed at pH 3.5, 5.0, 5.6, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.3, respectively. In this process, the inventors found that UBNS4E12-II protein was used for ion-exchange chromatography at each pH above, unlike normal protein, and S-Sepharose (Pharmacia) gel and Q-sepharose (Q- Sepharose, Pharmacia) gel was found not to be adsorbed at all. In addition, in the S-200 gel filtration chromatography with a fractionation protein range of 250K, UBNS4E12-II, which has a monomer size of about 32K, does not elute in the theoretical dissolution range, and proteins of 250K or more are eluted without selectivity. Eluted). From the above facts, the inventors have found that the UBNS4E12-II protein exists in the form of a monomer or more. Therefore, the inventors of the present invention provide high purity through cell disruption and removal of soluble protein, protein inclusion body recovery and washing, inclusion body lysis, ion exchange chromatography, protein precipitation and washing with ammonium sulfate, gel filtration chromatography and two dialysis processes. A method for recovering UBNS4E12-II was developed, and the density of the purified UBNS4E12-II was measured to confirm that the protein was present in the form of particles.

An object of the present invention is to provide a method for efficiently purifying UBNS4E12-II, an epitope fusion protein of the Korean type hepatitis C virus, which is present in the form of particles.

Hereinafter, the present invention will be described in detail.

Cell precipitates expressing KHCV UBNS4E12-II protein are treated by ultrasonication and pulverized, followed by centrifugation to remove soluble proteins as supernatants and to obtain insoluble precipitate inclusion bodies. The inclusion body is washed with a buffer solution containing ethylenediamine tetraacetic acid (EDTA) and phenyl methyl sulfonylfluoride (PMSF) to remove contaminating proteins recovered with inclusion body in this step.

The inclusion body recovered and washed above can be dissolved using urea. The amount of urea to be used should be an amount capable of sufficiently dissolving the inclusion body, in the present invention is used 6 to 10M, preferably 8M urea solution.

The supernatant obtained by centrifugation of the inclusion body solution is then subjected to ion exchange chromatography. The ion exchange chromatography step can use cation exchange chromatography anion exchange chromatography as a purification step to adsorb other E. coli-derived proteins except the UBNS4E12-II protein to the resin of the ion exchange chromatography and elute the UBNS4E12-II protein. have. For purification of the UBNS4E12-II protein, S-sepharose is preferred as the resin of the cation exchange chromatography and cu-sepharose is used as the resin of the anion exchange chromatography. When performing cation exchange chromatography, the buffer may have a pH in the range of 3.5 to 6.0, with pH 4.0 being preferred. When anion exchange chromatography is performed, the pH of the buffer solution may be 9.0 to 10.5, and preferably pH 10.3. As a buffer of the buffer solution, citric acid is preferable for cation exchange chromatography, ethanolamine and cyclohexylamino propanesulfonic acid (CAPS) can be used for anion exchange chromatography, and CAPS is preferable. The urea concentration in the buffer solution is 6M to 8M, preferably 6M.

To apply the protein solution eluted from ion exchange chromatography to the gel filtration chromatography step, the volume of the solution must be reduced by at least 10 times. In general, an ultrafiltration concentration method is used to concentrate the protein solution, but UBNS4E12-II protein is highly adsorbed on the membrane when it is concentrated. Therefore, in the present invention, a precipitation method is used in which the protein is precipitated using ammonium sulfate and then dissolved in an appropriate volume. The amount of ammonium sulfate that can be used is at least 15% of the protein solution to be precipitated, preferably 15% is used. At this time, the pH of the solution is adjusted to 4.0 to 6.0 with 5N hydrochloric acid solution, it is preferable to adjust to pH 5.6.

The precipitated protein is recovered by centrifugation and dissolved in an appropriate amount of buffer solution. At this time, the concentration of urea in the buffer solution is 8M to 10M, preferably 8M, the pH of the buffer bath is 9.0 to 10.0, preferably pH 9.3.

Gel filtration chromatography steps are performed to obtain purified UBNS4E12-II protein with a final purity of at least 90%. As the resin, a resin having a fractionable range of 2000 K or more may be used, and Sepharose CL-4B (Pharmacia) is preferably used. The pH of the buffer solution used is 8.5 to 10.5, preferably pH 9.3. Further, the urea concentration in the buffer solution is 6M to 8M, preferably 6M.

The urea in the protein solution is then removed by dialysis to reshape the denatured protein. The pH range of the buffer used for dialysis is 9.0 to 10.0, preferably pH 9.5. In addition, ethanolamine or CAPS is used as a buffer.

Finally, secondary dialysis is performed to bring the pH of the protein solution closer to neutral, and the pH range of the buffer solution used for dialysis is 6.8 to 7.8, preferably pH 7.3. Also used as buffers are tris, phosphate buffered saline (PBS) and hydroxyethyl piperazine ethanesulfonic acid (HEPES), preferably HEPES. The density of the final purified protein can be measured to confirm that the UBNS4E12-II protein is in the form of particles.

The invention is explained in more detail based on the following examples. The following examples are illustrated to specifically illustrate the present invention and do not limit the scope of the present invention.

The composition of the buffer solution used in the examples below is as follows unless otherwise specified.

Buffer 1: 20 mM Tris, 1 mM EDTA, 1 mM PMSF, 150 mM NaCl, pH 8.0

Buffer 2: 8M urea, 20mM ethanolamine, 1mM EDTA, 1mM PMSF, pH 10.3

Buffer 3: 6M urea, 20mM CAPS, 1mM EDTA, 1mM PMSF, pH 10.3

Buffer 4: 6M urea, 20 mM ethanolamine, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, pH 9.6

Buffer 5: 20 mM ethanolamine, 1 mM EDTA, 1 mM PMSF, pH 9.6

Buffer 6: 20 mM HEPES, 150 mM NaCl, pH 7.3

Buffer 7: 6M urea, 20mM citric acid, 1mM EDTA, 1mM PMSF, pH 4.0

Example 1

Step 1: Cell disruption and removal of soluble proteins

After incubation of E. coli W3110 containing the vector ptrpH-UBNS4E12-II expressing the UBNS4E12-II protein (see ptrpH-UBNS4E12-II), patent application No. 94-1237), about 200 g of the cell precipitate (wet weight) in 200 ml of buffer Suspended by adding solution 1, and then sonicating in an ice bath (ultrasonic homogenizer 4710: Cole-Parmer, USA) for about 2 minutes at 50% power and 50% duty-cycle to destroy cells. I was. The cell homogenate thus obtained was centrifuged at 10000 rpm for 30 minutes with a high speed centrifuge (Beckman J2-21M, Rotor JA14) to remove the supernatant soluble protein and obtain an insoluble precipitate inclusion body.

Step 2: washing insoluble precipitate

The insoluble precipitate obtained in step 1 was suspended in a homogenizer (heavy-duty homogenizer: Cole-Parmer, USA) by adding about 200 ml of the buffer solution 1, and then centrifuged at 10000 rpm for 30 minutes using a high-speed centrifuge. The inclusion body as a precipitate was recovered.

Step 3: Dissolution of Inclusion Body

The inclusion body obtained in step 2 was dissolved in 150 ml of buffer solution 2 at room temperature for about 1 hour. After dissolution, high-speed centrifugation (10000 rpm / 40 minutes) removed the undissolved precipitate and only the supernatant was taken.

Step 4: Anion Exchange Chromatography

The same amount of buffer 3 was added to 150 ml of the supernatant of step 3, and then passed through a cu-sepharose column (Pharmacia, XK 50/20) equilibrated with buffer 3 at a rate of 2 ml per minute.

Step 5: Precipitation and Recovery of Proteins with Ammonium Sulfate

450 g of ammonium sulfate was added to 300 ml of the protein solution passed through the cu Sepharose column in step 4, and the pH was adjusted to 5.6 using 6N hydrochloric acid solution. After leaving for about 10 minutes, high-speed centrifugation (10000 rpm / 30 minutes) gave a precipitate.

Step 6: Dissolution of Precipitate with Urea

30 ml of buffer 2 was added to the precipitate obtained in step 5 to dissolve the precipitate. After dissolution, high-speed centrifugation (10000 rpm / 30 minutes) gave a supernatant.

Step 7: Sepharose CL-4B Gel Filtration Chromatography

30 ml of the supernatant of step 6 was subjected to gel filtration chromatography at a rate of 2 ml per minute through a Sepharose CL-4B resin column (Pharmacia, XK 50/100) equilibrated with Buffer 4, and eluted protein was fractionated for each 10 ml. Collected into. These fractions were 15% SDS-PAGE to separately collect 80 ml of specific fractions containing pure UBNS4E12-II protein only.

Step 8: Primary Dialysis

The UBNS4E12-II protein solution collected in step 7 was placed in dialysis only (Spectrum, USA, molecular cut: 10000-12000), and then dialyzed in a container containing 4 L of buffer 5 for 12 hours to remove urea.

Step 9: Second Dialysis

The protein solution first dialyzed in step 8 was dialyzed again for 12 hours in a container containing 4L of buffer 6. In this way, about 100 mg of UBNS4E12-II protein was obtained, and the purified UBNS4E12-II protein was analyzed by SDS-PAGE.

15% SDS-PAGE of the crude product and the final purified UBNS4E12-II protein obtained in each step of the stagnation process is shown in FIG. In FIG. 2, column 1 is the protein of standard molecular weight, column 2 is the total protein recovered after cell disruption, column 3 is the supernatant obtained by centrifugation of the washed and dissolved inclusion body, and column 4 is the ion. The protein eluted from the exchange chromatography, column 5 shows the UBNS4E12-II protein eluted from the gel permeation chromatography.

Example 2 Anion Exchange Chromatography

After diluting by diluting the same volume of buffer solution 7 in the protein solution of steps 1, 2, and 3 of Example 1, 2 ml per minute in a cu-sepharose column (pharmacia: XK 50/20) equilibrated with the buffer solution 7 Passed at speed. The passed protein solution was SDS-PAGE and the result is shown in FIG.

In FIG. 3, the first column is the supernatant obtained by centrifuging the protein of standard molecular weight, the second column is washed and dissolved inclusion body, and the third column is the cu-sepharose ion exchange resin in step 4 of Example 1. The protein eluted directly from the fourth column, respectively, shows the protein eluted directly from the cue-sepharose ion exchange resin of this example.

Confirmation Example 1 Gel Filtration HPLC of Final Purified UBNS4E12-II Protein.

To confirm the homogeneity of the UBNS4E12-II protein passed through step 7 of Example 1, buffer 4 was used as the eluent and gel filtration HPLC was performed using a Bio-Gel SEC 50-XL column (BIO-RAD). Was performed. It was confirmed that the UBE1E2 protein purified from the chromatogram (FIG. 4) was homogeneously present.

EXAMPLE 2 Density Measurement of Purified UBNS4E12-II Protein

The density of particulate UBNS4E12-II was measured by ultrafast centrifugation with cesium chloride. 0.5 ml of the UBNS4E12-II protein solution obtained in step 9 of Example 1 was placed in a 20 ml ultra-high speed centrifuge prepared with cesium chloride to have a density of 1.13 to 1.45 and centrifuged for 4 hours (BECKMAN L8-80). High speed centrifuge: rotor Ti 70). After centrifugation, the solution in the container was recovered in each 0.5 ml fraction and the weight of each fraction was measured and the relative amount of UBNS4E12-II in the fraction was measured by enzyme immunoassay (ELISA, Applicant's prior patent application 92-10039). Was measured. As a result, the density of the UBNS4E12-II protein was determined to be 1.21 to 1.27 (FIG. 5). This result confirmed that the purified UBNS4E12-II protein was in the form of particles. The results are shown in FIG. 5, in which the horizontal axis represents the number of fractions and the vertical axis represents the density and absorbance of each fraction.

Confirmation Example 3: Secondary structure comparison of purified UBNS4E12-II protein before and after urea removal

The secondary structures of the UBNS4E12-II protein obtained in step 7 of Example 1 and the UBNS4E12-II protein obtained in step 9 were compared by CD. As a result, the UBNS4E12-II protein from which urea was removed was found to have a more regular secondary structure than in the presence of urea (FIG. 6).

Confirmation Example 4: Confirmation of adsorption of UBNS4E12-II protein to the ion exchange resin

In order to confirm the adsorption conditions for the ion exchange resin of UBNS4E12-II in the protein solution which passed through steps 1,2,3 of Example 1, whether the adsorption was carried out at the following pH using cu-sepharose and es-sepharose resin It was confirmed. As a result, it was confirmed that the UBNS4E12-II protein was not adsorbed at the following pH at which the general monomer protein could be adsorbed. Therefore, UBNS4E12-II protein is not adsorbed on the ion exchange resin, while other E. coli-derived contaminant proteins can be adsorbed on the resin and removed.

pH buffer

4.2 Citric Acid

5.6 acetic acid

5.8 L-histidine

7.1 bis-tris propane

7.2 Phosphate

7.6 triethanolamine

8.0 Tris

8.5 diethanolamine

9.3 Ethanolamine

Comparative Example 1: Comparison of the elution pattern according to the type of gel resin when performing gel filtration chromatography

The protein solution, which passed through step 6 of Example 1, was packed into a gel filtration column (Pharmacia, XK 50/100), each filled with S-200 and Sepharose CL-4B and equilibrated with Buffer 4, and their elution forms were compared. .

Figure 7-A shows that the UBNS4E12-II protein is eluted in the void part in the elution form using S-200, Figure 7-B is the elution form using Sepharose CL-4B. It is shown that the UBNS4E12-II protein elutes in the elution range of this resin. As a result, it was confirmed that the UBNS4E12-II protein was present at a size of 250 KD or more.

Comparative Example 2: Confirmation of pH Condition in Ammonium Sulfate Precipitation

After adjusting the pH of the protein solution obtained in step 4 of Example 1 with 1N hydrochloric acid, the pH of the protein was precipitated by adding ammonium sulfate to 15% of the solution volume. FIG. 8 shows the results of SDS-PAGE of each sample. The first column shows the sample of standard molecular weight protein, the second row shows the protein before precipitation, the third row shows the precipitate formed at pH 10.3, and the fourth row shows the pH. The precipitate formed at 9.0, the fifth column is the precipitate formed at pH 8.0, the sixth column is the precipitate formed at pH 7.0, the seventh column is the precipitate formed at pH 6.0, the eighth column is the precipitate formed at pH 5.0, Row 9 shows the precipitate formed at pH 4.0, respectively. From the results of FIG. 8, it was confirmed that UBNS4E12-II protein was precipitated by ammonium sulfate at pH 6 or below.

Comparative Example 3: Determination of the amount of ammonium sulfate used in the precipitation of ammonium sulfate

The protein solution obtained in step 4 of Example 1 was adjusted to pH 5.6 with 1N hydrochloric acid, and then various amounts of ammonium sulfate were added to confirm precipitation of the protein. Each sample was shown in FIG. 9 by SDS-PAGE. In FIG. 9, the first column represents a sample of standard molecular weight protein, the second column represents a protein before treatment, and the third, fourth, fifth, sixth, seventh and eighth columns have a volume% of ammonium sulfate, respectively, 0,5,10, The precipitates at 15, 30 and 40 are shown.

From the results of FIG. 9, it was confirmed that the amount of ammonium sulfate capable of precipitating the UBNS4E12-II protein should be 15% or more based on the volume of the protein solution.

Claims (8)

In purifying the UBNS4E12-II protein, an antigen-determining site fusion protein of Korean hepatitis C virus expressed in Escherichia coli, A) disrupting cells to remove soluble proteins and recovering and washing inclusion bodies, B) recovering the supernatant by dissolving the inclusion body using urea and centrifuging; C) eluting the UBNS4E12-II protein by ion exchange chromatography using S-Sepharose or Q-Sepharose as the ion exchange resin, D) precipitating the protein with ammonium sulfate and dissolving with urea, E) gel filtration chromatography using a resin having a fractionable range of at least 2000 K and a buffer having a pH of 8.5 to 10.5, F) a primary dialysis step for removing urea, and G) a secondary dialysis step for pH adjustment of the protein solution. The method of claim 1, wherein the urea concentration of step b) is 6-8M. 2. The method of claim 1, wherein e-separose is used as the ion exchange resin of step c) and eluted using a buffer of pH 3.5-6.0 containing 6-8 M urea. 2. The method of claim 1, wherein elution is carried out using cu-sepharose as the ion exchange resin of step c) and using a buffer of pH 9.0 to 10.5 comprising 6-8 M urea and ethanolamine or CAPS as a buffer. The process of claim 1 wherein the final concentration in the solution of ammonium sulfate in the precipitation of step d) is at least 15% and the pH of the solution is from 4.0 to 6.0. The method according to claim 1, wherein the gel filtration resin of step e) is separated by Sepharose CL-4B, and eluted using a buffer containing ethanol amine or CAPS as a buffer. The method of claim 1, wherein the buffer used in step f) is a buffer of pH 9.0 to 10.5 comprising ethanolamine or cyclohexylamino propanesulfonic acid (CAPS) as a buffer. The method of claim 1, wherein the buffer used in step 4) is a buffer of pH 6.8 to 7.8 comprising Tris, Phosphate Buffered Saline (PBS) or hydroxyethyl pyreazine ethanesulfonic acid (HEPES) as a buffer.