Cloning And Expression Of A CDNA Encoding A Snake Venom Metalloproteinase

Cloning and Expression of a cl)NA Encoding a Snake VenomMetalloproteinaseAbstract:A cDNA clone of 987bp is amplified from the total RNA of Agkistrodon halys Pallas snake venom gland by RT-PCR. Deduced amino acid sequence indicates that HXL is composed of a partial open reading frame encoding 317 amino acid residues. It contains a zyrnogen domains a central metalloproteinase domain and a Arg-Gly-Asp (RGD) disintegrin domain. High conserved PKMCGVT motif and signature zincbinding motif HEXXHXXGXXH exist in zymogen domain and the catalytic region of metalloproteinase domain respectively. The RGD tripeptide sequence is suggested to bind to platelet receptor and inhibit the platelet aggregation. The predicted amino acid sequence shares high homologous with other snake venom hemorrhagic metalloproteinases and disintegrins. According to the deduced amino-acid sequence, we also discuss the possible processing mechanism of the precursor. We clone each functional domain into pET-22b to construct expression plasmids, then transform them into E.coli strain BL21(DE3), induce recombinant proteins with IPTG. HXL-A protein containing both metalloproteinase and disintegrin domains and HXL-B protein containing the metalloproteinase domain only are expressed in Escherichia coIl into inclusion bodies which appear nearly 20% of the total cell proteins and purified successfully with Ni-NTA affinity chromatography under denaturing conditions. Because it is a hard work to refold the denatured inclusion proteins correctly, especially for those which contain several disulfide bonds. the results?repeatability of some activities is not very perfect.