Cloning and functional expression of the mucrosobin protein, a beta-fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu).
A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5'-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His(41), Asp(84) and Ser(179) in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific beta-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquire a functional snake venom protein with therapeutic potential.
Guo
Y W
YW
Department of Biology and Anatomy
Chang
T Y
TY
Lin
K T
KT
Liu
H W
HW
Shih
K C
KC
Cheng
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014158:Transcription, Genetic; D017841:Trimeresurus / Q000235:genetics
D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D011993:Recombinant Fusion Proteins; C029748:Trimeresurus venoms; D005340:Fibrinogen; D012697:Serine Endopeptidases