CLONING AND FUNCTIONAL EXPRESSION OF A CDNA-ENCODING THE CATALYTIC SUBUNIT OF BOVINE ENTEROKINASE

Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by hig hly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. In this paper, we report the cloning and func tional expression of a cDNA encoding the catalytic domain (light chain ) of bovine enterokinase. The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of hom ology with a variety of mammalian serine proteases involved in digesti on, coagulation, and fibrinolysis. We have developed a novel expressio n method for the enzyme which utilizes the secretory leader and propep tide of the mammalian serine protease PACE fused to the enterokinase l ight chain amino terminus. Efficient cleavage of the paired dibasic am ino acid cleaving enzyme (PACE) propeptide was achieved by coexpressio n with human PACE or yeast KEX2. The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comp arable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic s ubstrate Gly(Asp)4-Lys-beta-naphthylamide. The recombinant single-chai n form of enterokinase was also capable of activating trypsinogen, ind icating that the specificity of the enzyme for its natural substrate i s retained even in the absence of the noncatalytic enterokinase heavy chain.