Optimize a system for obtaining humanized Fibroblast Growth Factor 2 (FGF-2h) with high yield using E.coli

• Grant number: 22/16419-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2023
• End date: December 31, 2023
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcelo Ferreira Marcondes Machado
• Grantee: Matheus Lucena Galhardo
• Host Institution: Instituto Nacional de Farmacologia (INFAR). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Fibroblast Growth Factor 2 (FGF-2 or bFGF) is a prototype derivative of the FGF family, which in turn is involved in tumor expansion and development. In the literature, there are several reports of its involvement with melanoma, due to its high expression by malignant cells, disease progression and poor prognosis. On the other hand, an anti-FGF-2 monoclonal antibody (mAb) named 3F12E7, developed by collaborators in the immunology group of Dr. Jane Zveiter de Moraes demonstrated high antigenic recognition by FGF-2 when referring to tumor growth and the appearance of recurrent melanoma metastases. Our research group is currently developing a project for the synthesis of nanobodies based on known mAbs, importing the amino acid sequences of characterized mAbs into the recognition regions (CDR's). The synthesis will be carried out using the overlapping PCR technique (OE-PCR) of oligonucleotides. Among the nanobodies to be developed, we have one in which the biochemical information of the CDR's is being imported from the VHH sequence of the 3F12E7 mAB. This project aims to obtain FGF-2h, by synthesizing the gene optimized for expression with high yield in an expression system using E. Coli, through OE-PCR. We hope to create a high-yield expression system for stable FGF-2h, since the FGF-2h molecules available on the market have high rates of instability due to the rapid loss of their protein structure.