Abstract
Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues.
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Acknowledgments
This work used the Cell Free Expression platform (Lionel Imbert) of the Grenoble center Tutorial (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with media from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank Ismael Feitosa Lima for excellent technical help. This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (1214/2011, 7737/14-9), Fundação de Amparo à Pesquisa do Estado de São Paulo (2013/07467-1) and from a dedicated grant from the Direction des Sciences du Vivant of the CEA.
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Menezes, M.C., Imbert, L., Kitano, E.S. et al. Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system. Amino Acids 48, 2205–2214 (2016). https://doi.org/10.1007/s00726-016-2255-7
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DOI: https://doi.org/10.1007/s00726-016-2255-7