Expression Of Bovine Enterokinase Light Chain Gene And Its Enzyme Activity Analysis

Bovine enterokinase light chain(EKL)is a highly specific protease that recognizes Asp-Asp-Asp-Asp-Lys sequence.It can hydrolyze polypeptides at the C-terminal of Lys and has extensive application value in protein production and bioengineering.Now,its current market price and the cost of extraction and separation is high,and the source of natural bovine enterokinase material is limited,which limits its further application.Based on this,this study uses genetic engineering methods to obtain high yield and enzymatic activity EKL through recombinant expression in vitro and optimization of expression conditions,thereby providing technical support for its development and application.1 Prokaryotic expression and enzyme activity analysis of bovine enterokinase light chain geneIncreasing the expression of EKL protein by replacing the ribosomal binding site on the pET-28a(+)vector.The RBS1 sequence upstream of the EKL gene(5’TTTTGTTTAACTTTAAGAAGGAGAGA-3’)was replaced for the first time with the RBS2 sequence(5’-AAAGGAGAAA-3’),and recombinant expression plasmids pET-28a(+)RBS1-EKL-His and pET-28a(+)-RBS2-EKL-His were constructed.After digestion identification and sequencing are correct,convert them into receptive state E.coli ArcticExpress(DE3).After IPTG induction,SDS-PAGE and Western-blot results showed that the fusion proteins RBS1-EKL-His and RBS2-EKL-His were mainly expressed in the form of inclusion bodies,and the expression level of RBS2-EKL-His was relatively high.The renaturation of the inclusion bodies and purification by affinity chromatography showed that the protein yield of RBS2-EKL-His increased by 55.20%compared to RBS1-EKL-His.The results of enzymatic activity showed that both of them could specifically cleave substrate proteins containing(Asp)4-Lys sequences.In summary,replacing RBS1 with RBS2 increased the expression of EKL with enzymatic activity.2 Eukaryotic expression and enzyme activity analysis of bovine enterokinase light chain geneAccording to the sequence of bovine EKL gene,primers were designed and synthesized.The EKL gene was amplified by PCR and cloned to the corresponding site of the eukaryotic expression vector pPIC9K,the recombinant expression plasmid pPIC9K-His-EKL was constructed.After correct digestion identification and sequence analysis,it was transformed into the histidine deficient Pichia pastoris host strain GS115.After screening and PCR identification,the results showed that the His-EKL gene was successfully integrated into the chromosomes of Pichia pastoris.Through optimization of induction conditions,the optimal fermentation conditions for expression of His-EKL in Pichia pastoris were determined as follows:induction temperature 30℃,initial pH of culture medium 5.0,methanol concentration 1%,induction time 72 h.Using affinity chromatography column for purification,SDS-PAGE and Western-blot results showed that the fusion protein His-EKL was successfully secreted and expressed in Pichia pastoris,with a purity of 98%and enzymatic activity.In this study,EKL was obtained in two protein expression systems.Compared with the E.coli prokaryotic expression system,by optimizing the fermentation conditions of Pichia pastoris,the secreted expression product EKL with higher expression amount and enzyme activity can be obtained,which is more conducive to the promotion and application of the laboratory and provides biological raw materials for reducing the cost of protein production.