PRODUCTION OF RECOMBINANT BOVINE ENTEROKINASE CATALYTIC SUBUNIT IN ESCHERICHIA-COLI USING THE NOVEL SECRETORY FUSION PARTNER DSBA

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 sequen ce (Asp)(4)-Lys. This stringent site specificity gives EK great potent ial as a fusion protein cleavage reagent. Recently, a cDNA encoding th e catalytic (light) chain of bovine enterokinase (EK(L)) was identifie d, characterized, and transiently expressed in mammalian COS cells. We report here the production of EK(L) in Escherichia coli by a novel se cretory expression system that utilizes E. coli DsbA protein as an N-t erminal fusion partner. The EK(L) cDNA was fused in-frame to the 3'-en d of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recogn ition 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 indisting uishable from the previously described COS-derived enzyme. Both forms of rEK(L) were capable of cleaving peptides, polypeptides and trypsino gen with the same specificity exhibited by the native heterodimeric en zyme purified from bovine duodena. Interestingly, rEK(L) activated try psinogen poorly relative to the native heterodimeric enzyme, but was s uperior in its ability to cleave artificial fusion proteins containing the (Asp)(4)-Lys recognition sequence.