Production by heterologous expression on Pichia pastoris and evaluation of the efficacy of a vascular endothelial growth factor and a serineprotease from snake venom, for the production of a new biopolimer with adhesive capacity

• Grant number: 23/11264-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2023
• Status: Discontinued
• Field of knowledge: Health Sciences - Pharmacy - Toxicological Analysis
• Principal Investigator: Eliane Candiani Arantes Braga
• Grantee: Isabela Gobbo Ferreira
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 21/11936-3 - Center for Translational Science and Biopharmaceutical Development, AP.CCD
• Associated scholarship(s): 24/14618-0 - Functional evaluation of CdtVEGF, the vascular endothelial growth factor from the Brazilian rattlesnake Crotalus durissus terrificus, BE.EP.PD

Abstract

The venoms of snakes are considered rich sources of components, including enzymes, peptides, and non-enzymatic proteins. Their various actions induce a wide range of effects, ranging from disturbances in the coagulation system to necrosis and inflammation. Moreover, there are reports of molecules present in snake venoms that increase vascular permeability, induce the proliferation and migration of specific cells, which can aid in the tissue repair process. These molecules are vascular endothelial growth factors (VEGFs), a class of homodimeric proteins with eight highly conserved cysteine residues in all six proteins of this class: VEGFs A, B, C, and D, known as endogenous VEGFs, VEGF-E identified in a viral genome, and VEGF-Fs identified in snake venoms. These molecules are structurally and functionally very similar, differing in their ability to bind and activate receptors for VEGFs expressed in different cells, such as monocytes, fibroblasts, and vascular endothelial cells. VEGF-Fs differ from endogenous VEGFs by having a heparin-binding region in the C-terminal portion of the molecule and by not having glycosylations. They are smaller proteins (between 20 and 30 kDa) and more soluble than VEGFs-A. VEGFs interfere in wound healing by inducing collagen deposition, vessel re-epithelialization, and inducing migration of vascular endothelial cells, leading to the growth of new vessels through angiogenesis reestablishment.Commercial fibrin sealant is a hemostatic and adhesive agent composed of concentrated fibrinogen and thrombin extracted from human blood and is expensive. Various types of sealants have been developed with the aim of replacing sutures, avoiding complications such as fistulas, tissue ischemia, or tearing of inflamed areas. The Center for the Study of Venoms and Venomous Animals (CEVAP) has developed a heterologous fibrin sealant, with pre-clinical and phase I and II clinical trials already completed. This sealant contains a serine protease purified from the venom of the snake Crotalus durissus terrificus, a cryoprecipitate rich in fibrinogen extracted from buffalo plasma, and the calcium chloride diluent, which polymerizes in situ. rCollineína-1 is a recombinant serine protease from the venom of Crotalus durissus collilineatus with fibrinogenolytic action. Therefore, it has the potential application for the development of a new biopolymer with adhesive capacity.The purification of toxins in their native form has a low yield, with consequent low recovery (for VEGF between 0.5 and 2% and for Collineína-1 between 1 and 2%). In other words, obtaining both proteins through heterologous expression will enable their production on a larger scale at a low cost. Thus, the current project aims to produce VEGF and collineína-1 through heterologous expression on an expanded scale, in the yeast system Pichia pastoris, aiming at the development of a new biopolymer with adhesive capacity and commercial scalability. Additionally, the project aims to perform PEGylation (modification of biomolecules by covalent conjugation with polyethylene glycol - PEG, a non-toxic and non-immunogenic polymer) of the VEGF and collineína-1 molecules to enhance their use as biopharmaceuticals, and to perform their structural, biochemical, and functional characterizations. The efficiencies of recombinant VEGF in enhancing cell migration (in vitro assays) and the new biopolymer in accelerating bone regeneration in an animal model will be analyzed. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

WIEZEL, GISELE A.; OLIVEIRA, ISADORA S.; REIS, MOUZARLLEM B.; FERREIRA, ISABELA G.; CORDEIRO, KALYNKA R.; BORDON, KARLA C. F.; ARANTES, ELIANE C.. The complex repertoire of Tityus spp. venoms: Advances on their composition and pharmacological potential of their toxins. Biochimie, v. 220, p. 23-pg., 2024-01-11. (23/11264-0, 21/11936-3, 20/13176-3, 22/08964-8, 23/01083-9, 23/10400-8)