title: Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.
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.
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, 05503-000, Brazil.
authors.forename: Milene C
authors.initials: MC
authors.lastname: Menezes
authors.affiliation: Institut de Biologie Structurale, University of Grenoble Alpes, 71 avenue des Martyrs, CS10090, 38044, Grenoble, France.|CNRS, IBS, 38044, Grenoble, France.|CEA, IBS, 38044, Grenoble, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Imbert
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, 05503-000, Brazil.
authors.forename: Eduardo S
authors.initials: ES
authors.lastname: Kitano
authors.affiliation: Institut de Biologie Structurale, University of Grenoble Alpes, 71 avenue des Martyrs, CS10090, 38044, Grenoble, France. thierry.vernet@ibs.fr.|CNRS, IBS, 38044, Grenoble, France. thierry.vernet@ibs.fr.|CEA, IBS, 38044, Grenoble, France. thierry.vernet@ibs.fr.
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, 05503-000, Brazil. solange.serrano@butantan.gov.br.
authors.forename: Solange M T
authors.initials: SM
authors.lastname: Serrano
mesh_terms: D000818:Animals; D017837:Bothrops; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D003435:Crotalid Venoms / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D045726:Metalloproteases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D003435:Crotalid Venoms; D011994:Recombinant Proteins; D045726:Metalloproteases; C543527:HF3 protein, Bothrops jararaca
keywords: Cell-free protein synthesis; Cys-rich proteins; Disintegrin-like/cysteine-rich; Recombinant protein expression; Snake venom metalloproteinase