Recombinant production of proteinases from Bothrops jararaca venom using eukaryotic cell-free protein synthesis systems

Abstract

Snake venom proteinases participate in snakebite pathology such as hemorrhage, inflammation, necrosis and hemostasis imbalance. Metalloproteinases (SVMPs) are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Serine proteinases (SVSPs) are synthesized as precursors containing a pro-peptide. Recombinant expression of the precursor of SVMPs and SVSPs has failed due to the instability of the polypeptide structure. Conversely, functional recombinant non-catalytic domains of SVMPs were obtained by prokaryotic expression systems. We recently showed 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 indicated that recombinant expression using prokaryotic-based cell-free synthesis is an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues. However, most venom proteinases are glycosylated proteins and their carbohydrate chains play an important role in their stability and activities, and the recombinant proteins obtained using prokaryotic-based cell-free synthesis are naturally not glycosylated. In order to overcome this issue and to gain a better understanding of the relationship between structure and function of proteinases present in B. jararaca venom, the aim of this study is to obtain the following proteins in recombinant form using eukaryotic cell-free protein synthesis systems: (i) The metalloproteinase HF3 and the serine proteinase PA-BJ, will be expressed using the 1-Step CHO High-Yield IVT System, a mammalian in vitro translation system based on CHO cell lysates, in order to obtain sufficient quantity of proteins and in glycosylated form, for structural analysis; (ii) In parallel, CHO cell-derived translationally active lysates will be generated and adapted for in vitro reaction conditions for the expression of HF3 and PA-BJ in order to improve the yield, and to modulate the protein glycosylation process.