KR960013392B1 - Recovery process of protein inclusion bodies recombinantly produced by e. coli. - Google Patents

Abstract

The protein inclusion bodies expressed in E. coli is purified by centrifuging E. coli culture media at low temp., collecting its precipitate, suspending the ppt. in an acidic buffer which is containing an surfactant in order to suppress unstable factors, homogenizing, centrifuging at low temp., and re-collecting the ppt.(homogenizing step); suspending and strring the ppt. in a solution which is composed of 0.5-0.1% nonionic surfactant and 10-50mM EDTA, diluting with a solution contg. 0.05-0.2% surfactant and 100-500mM NaCl, centrifuging, and collecting the ppt. which is containing the protein inclusion bodies removed almost all impurities e.g. cell membranes etc.(NaCl washing step); finally resuspending the inclusion bodies in an adequate amount of distilled water, stirring violently, centrifuging, and collecting the highly pure inclusion bodies(washing step with distilled water).

Description

Recovery method of protein inclusion body expressed in E. coli

Figure 1 shows the results of sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) of the inclusion body recovery step by step when the cells are ground at acidic pH (4.5),

2 shows a comparison of the degradation rate over time of the intestinal calcination hormone under two different pH conditions (pH 7.4 and pH 4.5),

Figure 3 shows the manner in which the inclusion body is degraded by protease by SDS-PAGE.

The present invention relates to a method for recovering the inclusion body of the heterologous protein expressed in E. coli, more specifically, the heterologous product produced in the form of protein inclusion body in the recombinant E. coli (hereinafter referred to as recombinant E. coli) produced by genetic engineering techniques In the method for purifying proteins, the present invention relates to a method for purifying a protein comprising the steps of recovering the inclusion body of the protein in an economical and high degree of water by suspending and grinding the recombinant E. coli in a buffer buffer containing a surfactant.

Most of the heterologous proteins expressed in E. coli by genetic engineering techniques are formed into protein inclusion bodies, which are collections of proteins that appear as bright spots under a microscope, and the inclusion bodies of these proteins can be recovered from the cytoplasm and recovered. This effect on the overall protein yield is great. Since the protein inclusion body is unstable, it may be suddenly degraded by changes in the environment inside and outside the cell, and a considerable amount may be lost. For this reason, the following may be suggested.

First, a series of proteases present in E. coli are active under special circumstances, resulting in the breakdown of unproduced double proteins in normal host cells (Neidhart Frederic C., Escherichis coil and Salmonella typmurium, American Society). for Microbiology, 680-691 (1987): Chung CHand Alfred L. Goldberg, Pro. Natl. Acid. Sci. USA 78 (8), 4931-4935 (1987)).

Second, when the inclusion body is exposed to the outside of the cell and subjected to environmental changes (pH, temperature, ionic strength, etc.), depolarization occurs as a non-polar defect such as asparagine or glutamine polymerizes at a certain position in the denatured polypeptide. Deamidation, and some of these reactions are further progressed to break the peptide bonds of the protein into small pieces. It is known that the frequency is higher in conditions above this phenomenon neutral (pH 7.4) (Liu, D. T-Y., Trends Biotech. 10, 364-369 (1992)).

Korean Patent Application No. 92-4281 describes a method for expressing and purifying growth hormone in E. coli. According to him, the protein inclusion body is recovered by crushing the cells at neutral pH. This condition can easily provide these two factors so that a significant amount of inclusions can be lost.

As a result of research efforts to solve the above problems of the present inventors, it was found that by adjusting the pH of the buffer solution at the time of cell grinding to an acidic state, it is possible to increase the recovery yield of the protein inclusion body and the purification yield of the final protein to complete the present invention. Haga arrived.

The expected effects of grinding cells in an acidic buffer solution include the following.

Firstly, the activity of protease is mainly at pH 6-9, so that in the state below pH 6, it is possible to prevent the degradation of the desired protein by the second: second, protein deamination and subsequent occurrence at neutral or higher conditions Isolation can be prevented: Thirdly, a significant fraction of E. coli-derived water-soluble proteins can be modified to produce very fine precipitates, which are much more effective when separating very large protein inclusions and microparticles in relatively low resolution continuous centrifugation: However, when cells are pulverized at neutral pH, nucleic acid in the form of continuous polymer (especially DNA) in the cell is viscous, so it is not easy to handle them, but it is easy to handle them because of their precipitation at acidic pH. Much more effective: Fifth, the efficiency of continuous centrifugation after cell disruption It is very high and therefore economical as it can reduce subsequent encapsulation cleaning processes.

Hereinafter, the present invention will be described in detail.

The E. coli precipitate recovered by low temperature centrifugation of E. coli was suspended in an acidic buffer solution containing a nonionic surfactant to inhibit the destabilization tolerance of the protein inclusion body, followed by pulverization and recovery of the protein inclusion body by low temperature centrifugation. ). At this time, more than 97% E. coli is pulverized, E. coli-derived nucleic acid and protein precipitated as fine particles and the viscosity disappears, so the efficiency of subsequent centrifugation is increased.

The recovered inclusion body was suspended and stirred in a solution containing 0.5 to 0.1% of nonionic surfactant and 10 to 50 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.5 to 8.5), and 0.05 to 0.2% of nonionic surfactant and 100 After dilution with a solution containing 500 mM sodium chloride, centrifugation is performed to recover the inclusion body from which most residual cell membranes, nonprotein impurities, and other impurities are removed (sodium chloride washing process). Thereafter, the inclusion body is suspended again in an appropriate amount of distilled water, vigorously stirred, and centrifuged to recover a high purity inclusion body (distilled water washing process).

The low-temperature centrifugation as described above refers to centrifugation after cooling to lower temperature or by cooling to a stirrer. As the nonionic surfactant, Triton X-100 or Tween 80 may be used, and the acidic buffer solution according to the present invention may contain 10 to 50 mM sodium acetate, and the pH may be 3.5 to 6. The pH is preferably 4 to 5. In strong acids, the protein inclusion body itself melts, making it difficult to separate, so it is not preferable to set the pH of the buffer solution to 3.5 or less. As a centrifuge, a continuous centrifuge (separator type BTPX 205, Alpha-Laval) can be used. Grinding is preferably a homogenizer, which is a continuous cell grinder, twice or more at a pressure of 600 to 700 bar at a flow rate of 8 L per minute.

Hereinafter, the present invention will be described in more detail with reference to Examples. Example is to carry out the protein inclusion body recovery of calcined hormone in accordance with the present invention and Comparative Example was experimented to quantify how much the protein inclusion body of calcined hormone is degraded by the enzyme according to the pH of the buffer.

In the following example, the present invention shows a process for recovering the haejangjang hormone inclusions from E. coli LUCK-BST-1E (Accession No. KFCC 10693) deposited on May 25, 1990, by the present applicant, the present invention It is not limited. It is apparent that the protein inclusion body recovery process of the present invention can be used for all proteins produced in the form of protein inclusion bodies in E. coli.

Example

E. coli (KFCC-10693) cultures containing sintered hormonal inclusion frames were obtained, which were cultured in a 450 L fermenter (initial volume 275 L, turbidity (O.D600) 80). This was transferred to a cooling stirrer and separated by a continuous centrifuge while gradually lowering the temperature to obtain a cell precipitate containing 927 g of inclusion bodies, to a final volume of 180 L in an acid buffer (10 mM sodium acetate, 0.1% Triton X-100, pH 4.5). Suspended and stirred.

The suspension was passed twice using a homogenizer (Apa-Laval) under a pressure of 650 bar, twice a minute at a flow rate of 8 L per minute, so that E. coli crushed the cell membrane and mixed well in a cooling stirrer to contain 919 g of protein inclusion body. The cell grinding liquid was obtained.

The pulverized liquid was separated by a continuous centrifuge, and tritonS-100 and EDTA were added to a precipitate containing 866 g of protein carrier to prepare a 50 L solution having a final concentration of 0.75% and 50 mM, respectively. Blinkman) vigorously exchanged for 30 minutes and then mixed with 130L 140mM sodium chloride solution, diluted to a final concentration of Triton-X-100 and sodium chloride in the solution to 0.2%, 100mM, respectively, and vigorously stirred with polytron again. This was continuously centrifuged to obtain a precipitate containing 820 g of protein inclusion body.

The precipitate was diluted with distilled water to a final volume of 100 L, stirred vigorously with polytron and then subjected to continuous centrifugation to obtain a pure purity precipitate containing 790 g of protein inclusion body.

The result of the process of recovering the inclusion body by the above method is shown in FIG. 1 as 15% soda dodecyl sulfate polyacrylamide gel electrophoresis (15% SDS PAGE).

The first column shows the total protein of the E. coli culture medium expressing the plastic growth hormone, and the second protein shows the total protein after the E. coli collected by continuous centrifugation of the culture solution in supernatant sodium buffer solution. The third row is the result of pulverizing E. coli suspension twice with a continuous cell mill, and the fourth row is a continuous centrifugation to recover the inclusion body. Row 5 is Triton X-100 washed with a solution containing EDTA and diluted with sodium chloride solution, column 6 is recovered by continuous centrifugation again and suspended and washed in distilled water. The seventh column is again suspended in distilled water to solubilize the final inclusion body precipitate obtained by continuous centrifugation.

Comparative example

500 ml of the protein suture containing Escherichia coli culture used in the Example was taken, and each 250 ml was separated by centrifugation (Induction Drive Centrifuge, Model J2-21M, EECKMAN Co., Ltd.) at 7500xg for 15 minutes to recover E. coli. Suspend the solutions in 250 ml of pH 7.4 buffer (10 mM Tris, 0.1% Triton X-100, 50 mM EDTA) and 250 ml of pH 4.5 (10 mM sodium acetate, 0.1% Triton X-100) at room temperature for 8 hours with a magnetic stirrer. Samples were taken at 1 hour intervals with stirring and analyzed by 15% SDS-PAGE.

The SDS-PAGE gel was dried and scanned with a densitometer (Video Densitometer, Model 620, BIO-RAD) to calculate the decomposition rate. In the Tris buffer at pH 7.4, about 40% of the inclusions were degraded within the first hour, while in sodium acetate buffer at pH 4.5, about 11% were degraded during the first hour.

In addition, the results of the degradation of the inclusion body by the method of 15% SDS-PAGE are shown in FIG. 3, FIG. 1 shows the arrow (a) to the normal position of the ectopic hormone in the 15% SDS-PAGE gel. In the second row, the protein inclusion body of the bovine sperm hormone is decomposed and appears at the position of the arrow (b) and below. Column 3 shows the E. coli total protein in the digestion mechanism.

Claims (3)

In the process of purifying the animal growth hormone produced in the form of protein inclusion body in recombinant E. coli, E. coli expressing the protein inclusion body is pulverized in an acidic buffer solution of pH 3.5 to 6.0 containing a surfactant to recover the protein inclusion body Purification method comprising the step. The method of claim 1, wherein the precipitate obtained after the grinding is mixed with a solution containing a surfactant and ethylenediamine tetraacetic acid, stirred, diluted with 100 to 500 mM sodium chloride, centrifuged, and the precipitate obtained is washed with distilled water and centrifuged again. Further comprising the step of separating. The method of claim 1 wherein the acidic buffer solution comprises acetate at a concentration of 10-100 mM.