Cloned factor C cDNA of the Singapore horseshoe crab, Carcinoscorpius rotundicauda and purification of factor C proenzyme

Abstract

Full-length and deletion subclones of cDNAs for Factor C of Carcinoscorpius rotundicauda are provided. These cDNAs have been cloned into .lambda.gt 22 and pGEM 11Zf(+). Further manipulations of the 5' and 3' ends of these cDNAs have been carried out, and these cDNAs have been further subcloned into other expression vectors such as pGEMEX-1, pET 3b, and the yeast shuttle vectors YEpsec 1 and pEMBLyex 4, and pPIC 9 and pHIL D2. Also provided are host cells transformed with expression vectors containing DNA molecules encoding proteins having Factor C-like enzymatic activity, methods of producing such proteins, methods for purifying Factor C zymogens, and methods for protecting Factor C zymogens from autoactivation by Gram negative bacterial endotoxin while the proenzyme is being purified and/or processed from amoebocyte lysates or from recombinant clones, or during storage or subsequent handling. This protection is afforded by the addition of 5-30% Me.sub.2 SO, which reversibly inhibits the Factor C zymogen.