Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

ABSTRACT

Human enterokinase (synonym enteropeptidase, EC 3.4.21.9) light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia coli codon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 15000 (enzymesubstrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104 U/mg.