Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)(4)-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage reagent. Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EK(L)) was identified, characterized, and transiently expressed in mammalian COS cells. We report here the production of EK(L) in Escherichia coli by a novel secretory expression system that utilizes E. coli DsbA protein as an N-terminal fusion partner. The EK(L) cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site. Active, processed recombinant EK(L) (rEK(L)) was generated from this fusion protein via an autocatalytic cleavage reaction. The enzymatic properties of the bacterially produced rEK(L) were indistinguishable from the previously described COS-derived enzyme. Both forms of rEK(L) were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena. Interestingly, rEK(L) activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)(4)-Lys recognition sequence.
