Purification and preparation process of recombinant staphylokinase mutant

A preparation process, a technology of staphylokinase, which is applied in the directions of biochemical equipment and methods, hydrolase, microorganisms, etc., can solve the problems of complex purification steps, low yield, endotoxin pollution, etc.

Inactive Publication Date: 2021-09-21
MABWELL (SHANGHAI) BIOSCIENCE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the amount of staphylokinase secreted is very small, and it is directly fermented by Staphylococcus aureus, it is easily polluted by the endotoxin produced by Staphylococcus aureus itself, so the yield is low and the purification steps are complicated

Method used

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  • Purification and preparation process of recombinant staphylokinase mutant
  • Purification and preparation process of recombinant staphylokinase mutant
  • Purification and preparation process of recombinant staphylokinase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Homogenate of Engineering Bacteria and Treatment of Homogenate

[0060] Since mSAK is a soluble protein expressed in the large intestine, resuspension, homogenization, centrifugation, etc. are required before chromatographic purification to release the target protein in the cells. Therefore, it is first necessary to compare the homogenization conditions and whether the post-homogenization treatment will affect the subsequent purification. Take 50g each of three parts of the same bacteria sludge, and perform homogenization and post-homogenization treatment according to the method in Table 1 respectively:

[0061] Table 1 Homogenization and post-homogenization treatment

[0062]

[0063] The three groups of samples were subjected to Chelating Ni2+ chromatography after homogenization and post-homogenization, and compared the influence of different processing methods on the chromatographic purity. The results are as follows figure 1 shown.

[0064] accordin...

Embodiment 2

[0065] Example 2: Determination of recombinant staphylokinase capture method and process optimization

[0066] After the homogenate treatment method is determined, the target protein needs to be extracted from the homogenate supernatant. Since the centrifuged supernatant after homogenization is relatively complicated, in addition to the released mSAK enzyme (target protein), there are also culture medium, secondary metabolites, protease released by cell rupture, etc., so a purification method that efficiently captures the target protein should be selected .

[0067] According to the common capture method of general protein products and the characteristics of mSAK being a fusion protein of Trx (the C-terminus of Trx is coupled with a His tag), the ammonium sulfate (As) fractional precipitation method and the Chelating Ni2+ metal chelation chromatography method were chosen to capture proteins for comparison. See Table 2 for details.

[0068] Table 2 Comparison of methods for r...

Embodiment 3

[0083] Embodiment 3: Chelating Ni2+ chromatographic process optimization

[0084] 3.1 Determination of elution conditions

[0085] According to the previous linear elution results, the optimal elution conditions were determined by sequential elution at 8%, 15%, 25% and 100%.

[0086] experiment procedure:

[0087] Equilibrium Buffer: 50mmol / L Tris-HCl + 0.3mol / L NaCl + 0.01mol / L imidazole pH7.5;

[0088] Elution Buffer: 50mmol / L Tris-HCl + 0.3mol / L NaCl + 0.5mol / L imidazole pH7.5;

[0089] Elution conditions: 8%, 15%, 25% and 100% elution in sequence;

[0090] Chromatographic column: XK 16 / 20 Chelating Ni2+ 20ml;

[0091] Loading amount: 10mg / ml

[0092] Gradient collection of each gradient elution peak, SDS-PAGE electrophoresis (results such as Figure 5 Shown), calculated yield and purity (results are shown in Table 3).

[0093] Table 3 Statistics of each elution peak yield and electrophoresis purity

[0094]

[0095] table 3, Figure 5 It shows that the electrop...

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Abstract

The invention provides a purification and preparation process of a recombinant staphylokinase mutant. The purification and preparation process comprises the following steps of: step 1, homogenizing engineering bacteria and treating homogenate; step 2, capturing recombinant protein; and step 3, refining by ion-exchange chromatography. The capturing of the recombinant protein in the step 2 comprises the following steps of: loading an engineering bacterium cell homogenate treatment solution to a nickel affinity column, eluting a target protein by changing the concentration of imidazole, and simultaneously adding 0.3 mol/L NaCl to reduce non-specific adsorption caused by the charge effect. The method is good in repeatability and high in yield, staphylokinase with the purity close to 100% can be obtained, and the method is suitable for large-scale production in the pharmaceutical industry.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a process for the purification and preparation of recombinant staphylokinase mutants, in particular to the homogenization, affinity purification and ion exchange purification of recombinant staphylokinase mutants for soluble expression of recombinant staphylokinase mutants. body method. Background technique [0002] Staphylokinase (Sak), Sak is a proteolytic enzyme secreted by lysogenic Staphylococcus aureus. Since the amount of staphylokinase secreted is very small, and it is directly fermented by Staphylococcus aureus, it is easily polluted by the endotoxin produced by Staphylococcus aureus itself, so the yield is low and the purification steps are complicated. In the early 1980s, Sako T et al. extracted the S-￥-C phage fragment containing the staphylokinase gene from Staphylococcus aureus, connected the staphylokinase gene to the Pbr322 body by enzyme digestion, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N1/21C12R1/19
CPCC12N9/52C12N1/20
Inventor 田赵源范敏陆游于东安游猛谭小钉梅菲王宇钱子俊
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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