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 (hEK L) gene was designed and artificially synthesized with built-in codon blas towards Escherichia coli codon preference. The synthetic hEK L gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEK L 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-hEK L at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEK L was approximately seven times that of bovine enterokinase catalytic subunit (EKMax™, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEK L was with specific activity of 1.4×10 4 c U/mg.