Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSP12 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSP12 had a C-terminal hexahistidine tag attached to the GmSP12 sequence, rtSP12 was extended with GluAlaAla at the N-terminus, and rfSP12 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfS12 was O-glycosylated with a trimannosyl or hexamannosyl. The native inhibitor was active slightly on trypsin and highly on subtilisin and protemase K. The extended C-terminus in rhSP12 and rfSP12 enhanced activity on the two latter enzymes and rendered rfSP12 active on clastase and pronase, but abolished the inhibition of trypsin. The glycosylation of rfSP12 reduced its inhibitory activity to a level comparable with the native inhibitor. The rtSP12 with tripeptide extension at the N-terminus and no C-terminal modification was clearly less active than the native inhibitor. None of the tested compounds inhibited a-chymotrypsin and the non-serine protemases. (c) 2005 Elsevier Inc. All rights reserved.