Expression，reflolding and preliminary characterization of recombinant snake venom metalloproteinases：Implication for the hemorrhagic mechanism

Two cDNAs encoding hemorrhagic snake venom metalloproteinase acutolysin A and non-hemorrhagic metalloproteinase(BR)were cloned into the expression vector pET-22b,respectively,and the corresponding two recombinant proteins,A-22b and BR-22b,were produced in inclusion bodies in E.coli BL21(DE3).The reombinant proteins were then subjected to solubilization,purification and refolding in vitro.A-22b showed hemorrhagic activity and fibronectin.Natural autolysin A had both hemorrhagic activity and proteolytic activity toward these substrates.BR-22b showed the proteolytic activities toward fibrinogen,but no hemorrhagic activity.In addition,two chimeric genes,C1 and C2,were constructed and cloned into pET-22b,and the corresponding recombinant proteins,C1-22b and C2-22b,were also expressed in inclusion bodies.C1-22b involved N-terminal 110 amino acidos of BR and C-terminal 95 amino acids of acutolysin A,while C2-22b contained N-terminal 108 amino acids of acutolysin A and C-terminal 112 amino acids of BR. The biological activities of A-22b and BR-22b,respectively.Our results suggested that N-terminal major subdomain of a snake venom metalloproteinase might play a key role in hemorrhagic activity and have an appreciable effect on the selectivity for protein substrates.

Expression, refolding and preliminary characterization of recombinant snake venom metalloproteinases: Implication for the hemorrhagic mechanism

Two cDNAs encoding hemorrhagic snake venom metalloproteinase acutolysin A and non-hemorrhagic metalloproteinase (BR) were cloned into the expression vector pET-22b, respectively, and the corresponding two recombinant proteins, A-22b and BR-22b, were produced in inclusion bodies in E. coli BL21(DE3). The recombinant proteins were then subjected to solubilization, purification and refolding in vitro. A-22b showed hemorrhagic activity but no detectable proteolytic activities toward fibrinogen and fibronectin. Natural acutolysin A had both hemorrhagic activity and proteolytic activity toward these substrates. BR-22b showed the proteolytic activities toward fibrinogen, but no hemorrhagic activity. In addition, two chimeric genes, C1 and C2, were constructed and cloned into pET-22b, and the corresponding recombinant proteins, C1–22b and C2–22b, were also expressed in inclusion bodies. C1-22b involved N-terminal 110 amino acids of BR and C-terminal 95 amino acids of acutolysin A, while C2–22b contained N-terminal 108 amino acids of acutolysin A and C-terminal 112 amino acids of BR. The biological activities of C2–22b and C1–22b were similar to those of A-22b and BR-22b, respectively. Our results suggested that N-terminal major subdomain of a snake venom metalloproteinase might play a key role in hemorrhagic activity and have an appreciable effect on the selectivity for protein substrates.