Recombinant expression of canecystatin 5 (CaneCPI-5) in Pichia Pastoris: codon optimization and lyophilization tests

• Grant number: 16/14246-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2016
• End date: June 30, 2018
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Flavio Henrique da Silva
• Grantee: Patricia da Silva Lima
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil

Abstract

Cystatins are cysteine-peptidases reversible inhibitors proteins that act by competitive inhibition, blocking the proteolytic activity. The plant's cystatins, called phytocystatins, are one of the families of the cystatin superfamily. From the genome of sugarcane project (SUCEST) data, our laboratory has identified and conducted the heterologous expression of six sugarcane cystatins. The CaneCPI-1, the most studied of them, was used in the production of transgenic sugarcane that overexpress and harms the development of insect-pest larvae Sphenophorus levis which it fed. On the other hand, CaneCPI-4 was used in studies evaluating its potential as a cancer drug, being able to attenuate the invasive ability of breast cancer cells in Vitor, and inhibit the development of melanoma cells in vivo. The canecystatin CaneCPI-5, study object of this project, has already proven to be a good inhibitor of human cathepsin L, S. levis rSl-CathL, and also of human cathepsin B. Therefore, in future, studies can be conducted with this protein for producing transgenic plants as well as for therapeutic purposes. However, in these studies, the development of protein expression and preservation systems is necessary. This project will be developed an expression system for CaneCPI-5 with codon optimization in Pichia pastoris, as well as lyophilization of recombinant protein assays, in order to obtain a large amount of active protein by a long storage period.