Cloning,Expression And Purification Of Recombinant Bovine Enterokinase And Study On The Determination Of Activity

Enterokinase is a serine protease.Due of its relatively high specificity and wide activity range,bovine enterokinase has been widely used in genetic engineering besides playing an important role in research and development of protein drugs.In this paper,a E.coli BL21(DE3)strain that can express bovine enterokinase light chain protein was constructed,recombinant bovine enterokinase light chain protein was obtained after several steps of purification,then different enzyme specific activity detection methods were studied.Plasmid pET39b-BEK was constructed and transformed into E.coli BL21(DE3).The positive engineering strains were obtained after screening and identifying,and were fermented,cultivated and induced to express.Each 10 liter of fermentation broth can get about 250 g wet cells,according to the electrophoresis test,the target protein was mainly expressed in the form of inclusion bodies.Inclusion bodies were collected after breaking cells,5 g of wet inclusion bodies were taken for renaturation.then were purified by affinity chromatography,size exclusion chromatography and anion exchange chromatography.In the end,about 220 mg of target protein was obtained,indicating about 220 mg of purified target protein can be obtained from 1 liter of fermentation broth.The purified target protein was detected with a protein content of about 0.92 mg/mL,purity greater than 95%,and enzyme specific activity of about 55000 U/mg.Two-steps assay and fluorogenic substrate assay were used to test the enzyme specific activity of the purified products respectively.It was found that the former one was complex in operation and inaccurate in results,while the latter one was simple in operation and accurate in results,therefore,fluorescent substrate assay was selected as the enzyme specific activity detection method of product.The optimized reaction condition of the fluorescent substrate assay was 25? of incubation temperature and pH 8.0 of buffer solution.The fluorescence substrate assay was proved stable and reliable through methodological validation.Through comparing the fluorescent substrate assay with electrophoresis assay which using fusion protein as the substrate,the relationship between the enzyme specfic activity expressed by the fluorescent substrate assay and the enzyme specfic activity expressed by the electrophoresis assay was established to enable recombinant bovine enterokinase light chain protein can be used for digestion of the fusion protein.