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cDNA Cloning and Expression of Acutin,a Thrombin-Like Enzyme from Agkistrodon acutus

Biochemical and Biophysical Research Communications, ISSN: 0006-291X, Vol: 255, Issue: 2, Page: 412-415
1999
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Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence 〈VIGGVECDINEHRFL〉 was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for 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 residues, His 41, Asp 84 and Ser 179 ; and disulfide bridges, Cys 7 -Cys 139, 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 and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.

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