Bacterial expression of a snake venom metalloproteinase inhibitory protein from the North American opossum (D.virginiana).

R Marshall Werner, Lauren M Miling, Brianna M Elliott, Mitchell R Hawes, Jennifer M Wickens, Danielle E Webber

Toxicon : official journal of the International Society on Toxinology 2021 Apr 30

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A variety of opossum species are resistant to snake venoms due to the presence of antihemorrhagic and antimyotoxic acidic serum glycoproteins that inhibit several toxic venom components. Two virtually identical antihemorrhagic proteins isolated from either the North American opossum (D. virginiana) or the South American big-eared opossum (D. aurita), termed oprin or DM43 respectively, inhibit specific snake venom metalloproteinases (SVMPs). A better understanding of the structure of these proteins may provide useful insight to determine their mechanism of action and for the development of therapeutics against the global health concern of snake-bite envenomation. The aim of this work is to produce a recombinant snake venom metalloproteinase inhibitor (SVMPI) similar to the above opossum proteins in Escherichia coli and determine if this bacterially produced protein inhibits the proteolytic properties of Western Diamondback rattlesnake (C. atrox) venom. The resulting heterologous SVMPI was produced with either a 6-Histidine or maltose binding protein (MBP) affinity tag on either the C-terminus or N-terminus of the protein, respectively. The presence of the solubility enhancing MBP affinity tag resulted in significantly more soluble protein expression. The inhibitory activity was measured using two complementary assays and the MBP labeled SVMPI showed 7-fold less activity as compared to the 6-Histidine labeled SVMPI. Thus, the bacterially derived SVMPI with an unlabeled N-terminus showed high inhibitory activity (IC50 = 4.5 μM). The use of a solubility enhancing MBP fusion protein construct appears to be a productive way to express sufficient quantities of this mammalian protein in E. coli for further study. Copyright © 2021 Elsevier Ltd. All rights reserved.

Citation

R Marshall Werner, Lauren M Miling, Brianna M Elliott, Mitchell R Hawes, Jennifer M Wickens, Danielle E Webber. Bacterial expression of a snake venom metalloproteinase inhibitory protein from the North American opossum (D.virginiana). Toxicon : official journal of the International Society on Toxinology. 2021 Apr 30;194:1-10