Expression,Purification and Characterization of Bovine Enterokinase Catalytic Subunit in Escherichia coli

Enterokinase(EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence(Asp)_4-Lys. The DNA sequence encoding the light chain(catalytic subunit) of bovine enterokinase(GenBank Accession No.NM174439) was synthesized from the fresh bovine duodenal mucosa by reverse trascriptional polymerase chain reaction and cloned into plasmid pET39b downstream to the gene of fusion partner disulfide bond A immediately after the DNA sequence encoding enterokinase recognition site.The fusion protein disulfide bond A/bovine enterokinase light chain was expressed in Escherichia coli BL21(DE3) strain in soluble form.The soluble recombinant fusion protein can undergo catalytic cleavage and activation.The active recombinant bovine enterokinase light chain(rEK_L) was purified on nickel chelating chromatogram using His Binding Resin.The purified intact rEK_L was obtained 2.7-3.0mg from 1000 ml flask culture,and cleaved IL-11/MBP fusion protein with enterokinase recognition site.This investigation would be able to lay foundation for enterokinase activity research and further application of products on a large scale.