Heparanase II fusion protein and coding gene and expression method thereof

Abstract

The invention discloses a heparanase II fusion protein, and a coding gene and an expression method thereof. The fusion protein (named MBP-HepB) is a protein of a) or b), wherein a) is a protein consisting of an amino acid sequences shown from the 1st position to 1,118th position of a sequence 2 in a sequence table; and b) is a protein in which one or more amino acid is substituted, and / or deleted and / or added in the amino acid sequence of a sequence 2 in a sequence table and which has heparanase activity and derives from the a). The coding gene of the protein is also in the protection scope of the invention. The gene is introduced into the protein heparanase expressed in the recombinant escherichia coli, the enzyme activity reaches 118.4IU / L fermentation liquor, the expression quantity reaches 179mg / L fermentation liquor, and the specific enzyme activity reaches 0.66IU / mg. The one-step purification of the fusion protein can also be realized by affinity separation.

Description

technical field [0001] The invention relates to a heparanase II fusion protein, its encoding gene and its expression method in the field of genetic engineering and fermentation engineering. Background technique [0002] Heparinase (heparinase) is a class of polysaccharide lyase that acts on heparin (heparin) or heparan sulfate (heparan sulfate), found in many kinds of microorganisms, including Corynebacterium sp. (Gao Ningguo et al., Heparinase production Screening and fermentation conditions of bacteria, Acta Microbiology 1999 Vol.39:64-67), Sphingobacterium sp. 816), Bacillus subtilis (Wang Zhongyan, Screening of Heparanase-producing Bacteria and Research on the Properties of Crude Enzyme, Journal of Sichuan University (Natural Science Edition) 2002 Vol.39: 777-779), Bacillus circulans Bacillus circulans (Yasutaka Tahara et al., Purification and characterization of heparinase that degrades both heparin and heparin sulfate from Bacillus circulans BioSci.Biotechnol.Biochem....

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Problems solved by technology

[0006] However, the research on the heterologous recombination expression of heparanase II is very limited. So far, only Fu Wenbin and Su et al. have studied it in the literature.

Fu Wenbin and others used the pET expression system to realize the recombinant expression of heparanase II in Escherichia coli. SDS-PAGE electrophoresis results showed that there was a band of the target protein, but the expression level was very low, and inclusion bodies were easily formed. Soluble heparanase The activity is only 50U / L A 600 (Fu Wenbin, Yu Xiao et al., Cloning and Expression of Flavobacterium Heparanase II, Food and Drugs, 2007, Vol.9: 1-4); the activity of the expressed heparanase II in Su's study was improved, but Multi-step purification is required, resulting in relatively high purification costs, which is not conducive to industrial applications (Su H, blain F, Musil RA, Zimmermann JJF, Gu K, Bennett DC. Isolation and Expression in Escherichia coli of hepB and hepC, Genes Coding for the Glycosaminoglycan -Degrading Enzymes Heparinase II and Heparinase III, Respectively, from Flavobacterium heparinum.Appl.Environ.Microbiol.1996, 62:2723-2734)

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