Construction, The High-Density Fermentation And Purification Of Recombinant Enterokinase Catalytic Subunit In The Pichia Pastoris

Enterokinase (EK) , found widely in mammalian duodena , is a heterodimeric serine hydroltyic protease that can highly specifically hydrolyze trypsinogen into physiologically active trypsin. Ek is a dimer composed of a heavy peptide chain and a light peptide chain which are bound by a disulfide bridge, and its heavy chain anchors at the intestinal brush border membrane and its catalytic active site is only found in the light chain.EK recognizes amino acid sequence DDDDK of substrate and cleaves the peptide bond followed"K". Recently, EK has widely applied in gene engineer technique ,for example, in vitro cleaving fusion proteins secreted from Escherichia coli. The enzyme is extensively applied in gene engineer because of its high specificity, hydrolytic activity within the wide range of temperature and pH. Its recognition sequence lies entirely on the amino-terminal side of the scissile bond. This allows release of carboxyl-terminal fusion partners from fusion proteins without leaving unwanted amino acid residues on their amino termini. It is not expressed or purified because of the characterization of interest proteins when it is expressed in Escherichia coli. We added to the anterior guidance sequence before DDDDK and the foreign protein, and aimed to increase the yield of fusion protein expression and to easily purified.The cDNA of bovine enterokinase light chain was artificially synthesized based on the sequences in the GeneBank,and was inserted into the pPICZαA after validation to obtain plasmid pPICZαA/EKL, this was transmitted into Pichia pastoris SMD 1168H after linearization, thus the recombinant pichia pastoris strain was obtained followed by high-density fermentation of recombinant pichia pastoris strain, rEKL (recombinant enterokinase light chain, rEKL) purification, N-terminus sequences of EK examination and bioactivity assay. The results demonstrated that rEKL was highly expressed in the culture supernatant with high specificity and the amino acid sequences in N-terminus is consistent with the result of the other reported .