Purification And Application Of Bovine Enterokinase Catalytic Subunit (EK_L) Expressed From The Cloned CDNA Encoding EK_L

Enterokinase(EK)is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen into trypsin via a high specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage regent. Compared with proteases such as thrombin or factor Xa and chemicals such as CNBr or hydroxylamine, bovine enterokinase is an ideal choice because it can cleave with high specificity after the sequence (Asp)4-Lys, it can be tolerant to a wide range of conditions, and it allows any downstream fusion target protein to retain its native amino-terminus without leaving any unwanted amino acid residues on their amino termini. In this study, cDNA coded Chinese northern yellow bovine enterokinase catalytic subunite(EKL)was firstly cloned and expressed in E.coli and Pichia Pastoris by recombinant DNA techniques. The main results in this work are as follows:In the first place,the fragment of bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from a sold Chinese northern yellow bovine's duodenal mucosa, and then cloned into the pGEM?-T Easy cloning vector and sequenced．Compared with the sequence deposited in GenBank,the sequence of cDNA cloned in present study is correct.Expression in Prokaryotic Cells: By using DNA recombinant techniques, bovine EKL cDNA knocked out ending codes was inserted into expressive vector pET39b, and then transformed into E.coli BL21(DE3)pLysS. After optimization of condition , the yield of fusion protein DsbA-EKL-(His)8 which was first expressed in prokaryotic cell successfully accounted for more than 18% of total lysate proteins. Fusion protein was purified by Ni-NTA agarose affinity chromatography and autocatalytic cleaved by itself. Finally, the yield of purified, active rEKL-(His)8 from this method was 3.5mg from 1g of starting cell paste, and the specific activity of rEKL-(His)8 purified was more than 1.34×107U/mg.Expression in Eukaryotic Cells: Yeast Pichia pastoris expressive system was applied for expressing EKL. EKL cDNA was cloned into shuttle plasmid pPIC9, then the linearized recombinant plasmid pPIC9-EKL was transformed into Yeast Pichia GS115. The recombinant protein was obtained in fermentation supernatant. The yield of rEK was 32.8% of total supernatant protein. After separation and purification by STI resin affinity chromatography, the production of rEKL was about 10.9ug/mL fermented solution, which had a specific activity of 2.88×107U/mg.In this study, the autocatalytic cleavage of fusion protein DsbA-EKL-(His)8 and its protease action on fusion protein Trx-IL-11 were coupled for the first time. By one-step Ni2+-NTA affinity chromatography, the purity of rhIL-11 was more than 95%. This brand-new purification strategy would make more use of fusion proteins as a tool for recombinant protein production in our own country's biopharmaceutical industry.