Cloning And Expression Of Porcine Catalytic Subunit Of Enterokinase Gene In Pichia Pastoris And E.coli

Enterokinase, produced in mammalian duodenal epithelium, is a heterodimeric serine protease. It is composed by a heavy chain and a light chain and the ministry of the catalytic activity is located in the light chain. EK is a high specificity protease. It can recognize the specific peptides composed by four consecutive aspartate and a lysine and the next arbitrary amino acid(-DDDDK-↓-X- X represents any amino acid) and it can hydrolysis the peptide bond between lysine with any amino acid. Cutting the fusion protein by EK can ensure that other parts of the peptide bond in the fusion protein is not destroyed and the purpose peptide released from the cutting has the same N-terminal amino acid sequence with the wild-type peptide. So, EK is widely used as a cutting agent of fusion protein in protein expression system.Enterokinase is expensive on the market.This study is to prepare EK with the biological activity using genetic engineering methods. At present, there is many research on the bovine enterokinase. The study of the porcine enterokinase is rarely reported.So, taking into account the cost of production, expression and activity of the recombinant protein, etc, this study has a expression of porcine catalytic subunit of enterokinase, using using Pichia expression system and Prokaryotic expression system.First, EKL was cloned and was expressed in Pichia pastoris in this study. To prepare recombinant porcine catalytic subunit of enterokinase(EKL), we amplified EKL cDNA from porcine duodenal mucosa by RT-PCR according to porcine EKL gene sequence in GenBank. The amplified EKL gene was inserted into yeast shuttle vector pPIC9K down-stream ofα-factor signal peptide sequence by SnaB I and Not I sites to construct the recombinant secretory vector pPIC9K-EKL. The pPIC9K-EKL vector, linearized by Sal I, was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation. We selected His+-transformed methylotropic (His+, Mut+) yeast using histidine-absent medium containing dextrose (MD) or methanol (MM) as the only carbon source, and then screened the recombinant GS115 with multi-copy EKL genes by G418. The PCR test proved that EKL gene has been integrated into yeast chromosome. The secretive expression of EKL was performed under the induction of methanol in shaking flask culture. The expressed products were analyzed by SDS-PAGE and enzyme activity assay. The result showed that the recombinant protein with a molecular weight of 35 kDa was detected by SDS-PAGE in culture media. The recombinant protein showed the ability to convert trypsinogen into trypsin, which indicated that the recombinant EKL has the biological activity.Taking into account the cost of production and the convenience of expression, this test expressed EKL in E. coli. We amplified EKL by PCR. The amplified EKL gene was inserted into the prokaryotic expression vector pET-20b (+) by EcoRⅤand HindⅢsites to construct the C-terminal fusion His-tag prokaryotic expression plasmid pET-20b-EKL. The recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS. Then the positive clones were induced to express target protein by IPTG. Treating bacteria by sonication and centrifugation, we obtained the supernatant which we can used to have the analysis of SDS-PAGE and Western blot. The results showed that the prokaryotic expression vector of porcine EKL can mediate the recombinant porcine EKL expressed in E. coli BL21(DE3) pLysS. The recombinant porcine EKL, expressed in this study, can cut the fusion protein with the enterokinase site, indicating that the recombinant porcine EKL expressed in this study have the biological activity. This study has laid the foundation for further investigation of EKL application.