KALLISTATIN - A NOVEL HUMAN SERINE PROTEINASE-INHIBITOR - MOLECULAR-CLONING, TISSUE DISTRIBUTION, AND EXPRESSION IN ESCHERICHIA-COLI

We have recently purified a novel human serine proteinase inhibitor (s erpin), designated as kallistatin, which binds to tissue kallikrein an d inhibits kallikrein's kininogenase and amidolytic activities. In the present studies, we have cloned a full-length cDNA encoding kallistat in from human liver RNA by the polymerase chain reaction. The cDNA is 1284 base pairs in length and encodes 427 amino acid residues, includi ng a 26-residue signal peptide and a 401-residue mature peptide. The t ranslated amino acid sequence of kallistatin matches with the protein sequence and shares 44-46% sequence identity with human alpha1-antichy motrypsin, protein C inhibitor, corticosteroid-binding globulin, alpha 1-antitrypsin, thyroxin-binding globulin, and rat kallikrein-binding p rotein. Kallistatin is a new member of the serpin superfamily with a u nique reactive site P1-P1' of Phe-Ser. Four potential glycosylation si tes are found in the translated amino acid sequence of kallistatin. In a Southern blot analysis following reverse transcription and polymera se chain reaction, kallistatin was found to be expressed in human live r, stomach, pancreas, kidney, aorta, testes, prostate, artery, atrium, ventricle, lung, renal proximal tubular cell, and a colonic carcinoma cell line T84. A genomic Southern blot using the full-length kallista tin cDNA probe revealed simple banding patterns suggesting the gene en coding kallistatin is single-copied. The kallistatin cDNA encoding the mature peptide was expressed in Escherichia coli. The recombinant kal listatin forms an SDS-stable complex with I-125-human tissue kallikrei n and has a molecular mass of 40 kDa. The cloning of human kallistatin cDNA established the identity of the novel kallikrein inhibitor and i ts expression in a functional form in E. coli provides means for study ing its structure-function relationship through protein engineering.