Kallistatin: a novel human serine proteinase inhibitor. Molecular cloning, tissue distribution, and expression in Escherichia coli

• Published in: The Journal of biological chemistry 1993-11, Vol.268 (32), p.24498-24505
• Main Authors: CHAI, K. X, LI-MEI CHEN, CHAO, J, LEE CHAO
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Blotting, Southern; Blotting, Western; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; cDNA; Chromatography, Affinity; Chromatography, Ion Exchange; Cloning, Molecular; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; genes; Humans; Hydrolases; kallistatin; liver; man; Molecular Sequence Data; Multigene Family; nucleotide sequence; predictions; Rats; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA, Messenger - metabolism; Sequence Homology, Amino Acid; serine proteinase inhibitor; Serine Proteinase Inhibitors - genetics; Serine Proteinase Inhibitors - isolation & purification; Serine Proteinase Inhibitors - metabolism; serpins; Serpins - genetics; Serpins - isolation & purification; Serpins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(20)80553-5

We have recently purified a novel human serine proteinase inhibitor (serpin), designated as kallistatin, which binds to tissue kallikrein and inhibits kallikrein's kininogenase and amidolytic activities. In the present studies, we have cloned a full-length cDNA encoding kallistatin from human liver RNA by the polymerase chain reaction. The cDNA is 1284 base pairs in length and encodes 427 amino acid residues, including a 26-residue signal peptide and a 401-residue mature peptide. The translated amino acid sequence of kallistatin matches with the protein sequence and shares 44-46% sequence identity with human alpha 1-antichymotrypsin, protein C inhibitor, corticosteroid-binding globulin, alpha 1-antitrypsin, thyroxin-binding globulin, and rat kallikrein-binding protein. Kallistatin is a new member of the serpin superfamily with a unique reactive site P1-P1' of Phe-Ser. Four potential glycosylation sites are found in the translated amino acid sequence of kallistatin. In a Southern blot analysis following reverse transcription and polymerase chain reaction, kallistatin was found to be expressed in human liver, 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 kallistatin cDNA probe revealed simple banding patterns suggesting the gene encoding kallistatin is single-copied. The kallistatin cDNA encoding the mature peptide was expressed in Escherichia coli. The recombinant kallistatin forms an SDS-stable complex with 125I-human tissue kallikrein and has a molecular mass of 40 kDa. The cloning of human kallistatin cDNA established the identity of the novel kallikrein inhibitor and its expression in a functional form in E. coli provides means for studying its structure-function relationship through protein engineering.