Expression of a functional proteinase inhibitor capable of accepting xylose: Bikunin

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to hea vy chains via a chondroitin sulfate chain, forming inter-alpha -inhibitor a nd related molecules. Rat bikunin was produced by baculovirus-infected inse ct cells. The protein could be purified with a total yield of 20 mg/liter m edium. Unlike naturally occuring bikunin the recombinant protein had no gal actosaminoglycan chain. Endoglycosidase digestion also suggested that the r ecombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha (1)-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha (1 )-microglobulin-part of the alpha (1)-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recomb inant precursor. Both free bikunin and the precursor were also functional a s a substrate in an in vitro xylosylation system. This demonstrates that th e alpha (1)-microglobulin-part is not necessary for the first step of galac tosaminoglycan assembly. (C) 2001 Academic Press.