PRODUCTION OF A RECOMBINANT BOVINE ENTEROKINASE CATALYTIC SUBUNIT IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS

We describe the heterologous expression of a 26.3 kD protein containin g the catalytic domain of bovine enterokinase (EK(L)) in the methylotr ophic yeast Pichia pastoris. A highly active protein is secreted and g lycosylated, and it has the native amino-terminus of EK(L). The cDNA e ncoding EK(L) was cloned with the protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevi siae, The secreted EK(L) was easily purified from the few native prote ins found in the P. pastoris fermentation supernatant, using ion excha nge and affinity chromatography, The yield of the purified EK(L) was 6 .3 mg per liter of fermentation culture, This is significantly higher than previous reports of expressions in E. coli and COS cells, The abi lity of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)(4)-Lys recognition sequence all ows regeneration of native aminoterminal residues of recombinant prote ins, Its application is demonstrated by the removal of thioredoxin (Tr xA), and polyhistidine fusion partners from proteins of interest.