THE CORN INHIBITOR OF ACTIVATED HAGEMAN-FACTOR - PURIFICATION AND PROPERTIES OF 2 RECOMBINANT FORMS OF THE PROTEIN

A cDNA clone that encodes the 14-kDa bifunctional inhibitor from corn seeds (L. Wen ct al., Plant Mel. Biol. 18, 813-814, 1992) has been exp ressed in Escherichia coli after being incorporated into the pT7 expre ssion vector. This inhibitor protein, referred to as CHFI (for the cor n inhibitor of activated Hageman factor) or as the popcorn inhibitor, is an important tool for specific inhibition of human activated Hagema n factor (activated forms of coagulation Factor XII) and has been well characterized as isolated from corn seeds. Recombinant CHFI was expre ssed in E. coli in high levels but was insoluble. We solubilized the e xpressed protein by sonication in 5 M urea and 1% Triton X-100. Severa l steps of purification, culminating with reversed-phase HPLC, yielded pure, recombinant corn inhibitor in about 5% yield (about 1 mg per li ter of culture). The form with which we have worked most, 7N-CHFI, con tains 7 amino acid residues at its N-terminus that are encoded by the expression vector. Physical properties of this recombinant protein ind icate it has the expected mass and is properly folded. Functionally, 7 N-CHFI is indistinguishable from the inhibitor isolated from corn seed s in its inhibition of porcine trypsin, human beta-Factor XIIa, failur e to inhibit human plasma kallikrein, and its inhibition of an insect alpha-amylase. A second recombinant form, (4N-11)-CHFI, which lacks 11 residues from the corn inhibitor's N-terminus, is indistinguishable f rom 7N-CHFI in its pattern of inhibition of the three test proteinases but is inactive against the insect alpha-amylase. This suggests that the N terminal region of 7N-CHFI forms at least part of the protein's site of interaction with cu-amylase. (C) 1998 Academic Press.