A novel lysine-specific endonuclease and its preparation method

Abstract

The invention discloses novel lysyl endopeptidase. An enzyme core peptide is connected with histidine tags through an appropriate flexible linker peptide, so that nanogram residual enzyme in a samplecan be detected by an ELISA (enzyme-linked immunosorbent assay) method; and total activity of wild lysyl endopeptidase is ensured. At the same time, the invention further provides a preparation methodof the lysyl endopeptidase. Compared with a gene engineering modification and fermentation technology, the preparation method has the benefit of high yield; a recovery amount of 857mg novel lysyl endopeptidase can be obtained after purification of 1L fermentation liquid; and the method has an industrial potential.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel lysine-specific endonuclease and a preparation method thereof. Background technique [0002] Lysyl endopeptidase (Lysyl endopeptidase, EC 3.4.21.50) is also known as lysyl endopeptidase, 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. Lysine-specific endonuclease 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] Lysine-specific endonuclease also has the following notable 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...

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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 lysine-specific endonucleases, but the yield is not significantly improved, and the specific activity of recombinant lysine-specific endonucleases is increased. 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 lysine-specific endonucleases in drugs

High-performance liquid chromatography is used to detect the residues of lysine-specific endonuclease in intermediates and raw materials, and the detection limit can only reach the μg level, and the self-degradation band of lysine-specific endonuclease cannot be detected; Protein electrophoresis can detect lysine-specific endonuclease self-degradation bands, but the detection limit can only reach the μg level; the development cost of lysine-specific endonuclease ELISA detection kits is high, and there is currently no commercial The optimized ELISA kit can be used to detect the residue of lysine endopeptidase

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