Abstract

Various opossum species are resistant to snake venoms due to the presence of antihemorrhagic and antimyotoxic acidic serum glycoproteins that inhibit several toxic venom components. Snake-bite envenomation is a global health concern, and a better understanding of these proteins may provide useful insight to determine their mechanism of action and for the development of therapeutics Two identical antihemorrhagic proteins, termed oprin or DM43, isolated from either the North American opossum (D. virginiana) or the South American bigeared opossum (D. aurita), inhibit specific snake venom metalloproteinases (SVMPs). The aim of this work is to produce a recombinant snake venom metalloproteinase inhibitor (SVMPI) similar to the above opossum proteins. Eight constructs including the genes for the production of the proteins DM43 and maltose binding protein (MBP) were created in Escherichia coli vectors. The resulting construct was produced with a 6-Histidine and maltose binding protein (MBP) affinity tag on either the C-terminus or N-terminus of the protein, respectively. Four of the eight constructs were then used in the optimization of the enzymatic cleavage reaction between tobacco etch virus (TEV) and the fusion protein MBP-DM43 created by the vector. This new construct replaces a Factor Xa cleavage site with a TEV cleavage site. The cleavage reaction for the first construct (Assembly #1) was monitored and the use of the TEV cleavage site appears to be an efficient way to cleave the DM43 from the MBP in the fusion protein in that construct. A series of illustrations were created to accompany the scientific history and lab processes regarding this research.