Production of recombinant interferon beta 1b protein at plataform cell-free protein synthesis

• Grant number: 18/15794-6
• Support Opportunities: Regular Research Grants
• Start date: March 01, 2020
• End date: February 28, 2023
• Field of knowledge: Health Sciences - Pharmacy
• Principal Investigator: Marco Antonio Stephano
• Grantee: Marco Antonio Stephano
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Biopharmaceutical and immunobiological products represent a growing share of the global pharmaceutical market, totaling US$ 228 billion in global sales in 2016. Around 400 million people worldwide are dependent of such proteins and, often, they are going to need for the rest of their lives. So far, the most employed biopharmaceutical production technology is cell-based expression. However, this system is usually associated with problems such as degradation of proteins due to the presence of endogenous nucleases or proteases, aggregation with inclusion bodies formation and loss of DNA template. Cell-Free Protein Synthesis (CFPS) platform has emerged as an alternative to in vivo expression. CFPS system overcomes the main bottlenecks of cell-based expression, allowing synthesis of proteins in the soluble form, production of cytotoxic proteins and membrane proteins, and ensures high yield and scalability. This project therefore aims to synthesize recombinant human interferon-²1b (rhINF-²1b), biopharmaceutical applied in multiple sclerosis treatment, in CFPS system. A vector design of rhINF-²1b will be performed by bioinformatics software, with T7 promoter region, ribosomal binding site, start and end codon gene, and a T7 terminator. There will be 5 groups: negative control (without DNA), positive control (pEXP5-NT / CALML3), pEXP5-CT / TOPO, pEXP5-NT / TOPO and custom DNA template. The TOPO vectors will be cloned, and the custom vector will be amplified by PCR to obtain more copies. Then the expression vectors will be placed in the cell-free reaction of the Thermo Fisher Scientific commercial kit for protein production. Detection analyzes will be performed by electrophoresis, Western blot, Bradford and biomolecule chromatography. To study protein conformation, mass spectrometry, circular dichroism and crystallography will be performed. (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)

CHIBA, CAMILA HIROMI; KNIRSCH, MARCOS CAMARGO; AZZONI, ADRIANO RODRIGUES; MOREIRA, ANTONIO R.; STEPHANO, MARCO ANTONIO. Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products. BIOTECHNIQUES, v. 70, n. 2, JAN 2021. (18/15794-6)