Novel lysyl endopeptidase and preparation method thereof

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...

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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

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