Expression of recombinant Chinese bovine enterokinase catalytic subunit in P-pastoris and its purification and characterization

(E)nterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins. cDNA encoding the catalytic subunit of Chinese bovine enterokinase (EKL) was amplified by PCR and then fused to the 3' end of prepro secretion signal peptide gene of cc-mating factor from Saccharomyces cerevisiae to get the alpha-MF signal-EKL-His(6) encoding gene by PCR. Then the whole coding sequence was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter and transformed GS115 methylotrophic strain of Pichia pastoris. Secreted expression of recombinant EKL-His(6) was attained by methanol induction and its molecular weight is 43 W. Because of the existence of His(6)-tag, EKL-His(6) was easily purified from R pastoris fermentation supernatant by using Ni2+ affinity chromatography and the yield is 5.4 mg per liter of fermentation culture. This purified EKL-His(6) demonstrates excellent cleavage activity towards fusion protein containing EK cleavage site.