Recombinant Expression of Human Enteropeptidase Light Chain in Pichia pastoris

Abstract

The catalytic light chain of the serine protease human enteropeptidase (enterokinase, hEKLC) was expressed as an active recombinant protein in the yeast Pichia pastoris. EKLC shows high degrees of specificity and selectivity for the amino acid sequences DDDDK~X and DDDDR~X, making it useful for separating fusion domains of recombinant proteins. The amino acid sequence of hEKLC was altered to remove N‐linked glycosylation sites by changing Asn to Gln; similarly, an Arg in a potential kexin protease cleavage site was changed to Gln to preclude proteolysis. The cDNA encoding hEKLC was codon‐optimized for expression in P. pastoris and commercially synthesized. Fusion to α‐mating factor targeted the protein for secretion. Active recombinant hEKLC (rhEKLC) was purified from fermentation medium by liquid chromatography using the enzyme's affinity for soybean trypsin inhibitor (STI). Western blot results confirm the identity of rhEKLC using an antibody to bovine enteropeptidase. Enzymatic activity was characterized via cleavage of the synthetic substrates z‐Lys‐SBzl, GDDDDK‐p‐nitroanilide, and GDDDDR‐p‐nitroanilide. Since the enzyme is secreted in active form, its expression is useful for testing P. pastoris expression media and it has proven useful for activating other proteases expressed as fusion proteins linked via DDDDK sequences. NIH grant R15HL091770.