A kind of novel lysyl endopeptidase and preparation method thereof

A lysyl peptide chain and endonuclease technology, applied in the field of genetic engineering, can solve the problems of low ion exchange chromatography, low expression level, high development cost of ELISA detection kit, etc., to reduce production cost and use less materials Effect

Active Publication Date: 2019-12-13
ZHUHAI JINBAIKANG BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2. In order to overcome the shortcoming of low expression level of wild bacteria, people use engineering strains such as Escherichia coli to prepare recombinant lysyl endopeptidase, but the yield is not significantly improved, and the specific activity of recombinant lysyl endopeptidase is on the contrary reduced by
[0007] 3. When using traditional lysyl-specific endonucleases in the field of biopharmaceuticals, it is impossible to use highly sensitive methods to detect the residues of lysyl endopeptidases in drugs
High-performance liquid chromatography was used to detect the residual amount of lysyl endopeptidase in intermediates and raw materials, and the detection limit could only reach the μg level, and the self-degradation band of lysyl endopeptidase could not be detected; Protein electrophoresis can detect the lysyl endopeptidase self-degradation band, but the detection limit can only reach the μg level; the development cost of the lysyl endopeptidase ELISA detection kit is high, and there is currently no commercial The optimized ELISA kit can be used to detect the residue of lysine endopeptidase
[0008] 4. The conventional purification method of lysyl endopeptidase zymogen uses affinity chromatography instead of ion-exchange chromatography with lower cost

Method used

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  • A kind of novel lysyl endopeptidase and preparation method thereof
  • A kind of novel lysyl endopeptidase and preparation method thereof
  • A kind of novel lysyl endopeptidase and preparation method thereof

Examples

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Effect test

Embodiment 1

[0047] A gene encoding a novel lysyl-specific endonuclease (SEQ ID NO: 1) was artificially synthesized, wherein the flexible connecting peptide was selected as GGGGSGGGGS, and the continuous arginine was selected as two consecutive arginines. Construct genetically engineered strains, high-density culture, and enzyme activity detection and analysis according to the above methods. The enzyme activity of lysyl endopeptidase in the sample was detected to be 2280AU / L.

[0048] Take 1mL fermentation broth, and use SDS-PAGE electrophoresis to detect, and the detection results are as follows: figure 2 As shown, the results show that the molecular weight of the novel lysyl endonuclease is 29.8KDa, which is consistent with the theoretical value of 29814.46Da.

Embodiment 2

[0050] A gene encoding a novel lysyl-specific endonuclease (SEQ ID NO: 2) was artificially synthesized, wherein the flexible connecting peptide was selected as GGGGSGGGGS, and the continuous arginine was selected as four consecutive arginines. Construct genetically engineered strains, high-density culture, and enzyme activity detection and analysis according to the above methods. The enzyme activity of lysyl endopeptidase in the sample was detected to be 953AU / L.

[0051] Compared with Example 1, this example differs only in the number of continuous arginines, and the comparison of its enzyme activity shows that the continuous arginines are selected as 2 continuous arginines to keep the lysyl peptide The effect of endonuclease enzyme activity is better.

Embodiment 3

[0053] Preparation of novel lysyl endopeptidases:

[0054] Inoculate the novel lysyl endopeptidase (SEQ ID NO: 1) genetically engineered strain into a 20L fermenter containing 9L of fermentation medium, carry out fermentation and culture according to the above-mentioned high-density fermentation method, and remove the tank when the expression level reaches the highest . The fermentation broth was homogenized once with a high-pressure homogenizer at a pressure of 90MPa, loaded onto a UniGel 80SP chromatographic column, balanced with a balance solution (20mM trisodium citrate, pH4.0), and buffered with a buffer solution (20mM PBS, pH7. .6, the balance being water) for elution, collecting and obtaining the pure product of the novel lysyl endopeptidase, and vacuum freeze-drying to obtain the freeze-dried powder of the novel lysyl endopeptidase. According to the method described in Example 1, the specific activity of the novel lysyl endopeptidase was 2.42 AU / mg.

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Abstract

The invention discloses novel lysyl endopeptidase. A suitable flexible linker peptide is connected to an enzyme core peptide, a histidine label and an arginine tail end, while the original enzymatic activity is maintained, nanogram-level residual enzyme in a sample is detected by an ELISA method, and furthermore, novel lysyl endopeptidase sterling is obtained by purification of an ion exchange layer. The invention further provides a preparation method of the lysyl endopeptidase, after 1L of fermentation broth is purified, the recycling amount of 107 mg of the novel lysyl endopeptidase sterlingcan be achieved, and the novel lysyl endopeptidase has industrial potential.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel lysyl endopeptidase and a preparation method thereof. Background technique [0002] Lysyl endopeptidase (Lysyl endopeptidase, EC 3.4.21.50) is also known as lysine-specific endonuclease, Achromobacter protease I, lysine endopeptidase, lysyl endopeptidase, lysine Acid C-terminal endonuclease, Lys-C endonuclease, is a serine protease originally discovered and isolated from soil bacteria by Masaki et al. Lysyl endopeptidase is highly specific and can specifically cleave the peptide bond at the carboxy-terminal of lysine residues and S-aminoethylcysteine ​​residues in the peptide chain. [0003] Lysyl endopeptidase also has the following remarkable features: 1) 10 times higher activity than bovine trypsin; 2) wide pH tolerance (pH8.5-10.5); 3) stronger surface activity agent tolerance, such as maintaining full enzyme activity in a solution containing 4mol / L urea or 0.1% S...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/52C12N15/70
CPCC12N9/52C12N15/70C12Y304/2105
Inventor 赖红星马文柱夏玉平姚元锋雷春红肖拥军罗湘冀
Owner ZHUHAI JINBAIKANG BIOLOGICAL TECH CO LTD
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