cDNA cloning and expression of acutin, a thrombin-like enzyme from Agkistrodon acutus

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom i n three steps by DEAE-Sepharose CL-GB, Superose 12 column on FPLC and Mono- Q column chromatographies. Its first 15 N-terminal amino acid residues sequ ence [VIGGVECDINEHRFL] was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR, Determination of its nucleotide s equence allowed elucidation of the amino acid sequence of mature peptide fo r the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic r esidues, His(41), Asp(84) and Ser(179); and disulfide bridges, Cys(7)-Cys(1 39) Cys(26)-Cys(42), Cys(74)-Cys(231), Cys(118)-Cys(185), Cys(150)-Cys(164) , Cys(175)-Cys(200) The recombinant acutin has been expressed in E, coli an d purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginin e-esterase. (C) 1999 Academic Press.