Assessment of solubility and stability of a recombinant serine protease from Crotalus durissus collilineatus in its PEGylated and non-PEGylated forms

• Grant number: 24/02846-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: July 01, 2024
• End date: November 30, 2025
• Field of knowledge: Health Sciences - Pharmacy - Toxicological Analysis
• Principal Investigator: Eliane Candiani Arantes Braga
• Grantee: Isadora Lino Mendes
• 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

Abstract

The venom of the Crotalus durissus collilineatus snake is composed of several bioactive molecules, including collinein-1. This is a serine protease with the ability to interfere with hemostasis control mechanisms and is considered a promising alternative for the treatment of vascular and thrombotic disorders. The biopharmaceutical processes of heterologous expression and PEGylation have proven to be good tools for advancing the clinical use of this enzyme. However, further research is still needed on the changes that these modifications cause in the molecule. The aim of this project is to determine and compare the solubility and biological stability of a recombinant serine protease from Crotalus durissus collilineatus venom, named rCollinein-1, in its PEGylated and non-PEGylated form. Heterologous expression is performed using the yeast Pichia pastoris, and the recombinant protein is purified by immobilized metal ion affinity chromatography and cation exchange chromatography. The polymer mPEG-maleimide with a molecular mass of 5 kDa will be used for PEGylation and the reaction will be analyzed by reverse phase liquid chromatography on a C4 column. The solubility test is performed using the sonic shake method and the samples are analyzed by CLAE. Finally, the biological stability will beanalyzed by exposure to blood serum, acidic, basic and oxidizing media using dynamic light scattering, circular dichroism and enzyme activity. In this way, we hope to determine important factors for the characterization of rCollinein-1 and generate data on the usefulness of PEGylation in a recombinant SVSP, whose applicability has been little explored.