Development of DNA polymerases production process with high quality and processivity

• Grant number: 17/12334-1
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: December 01, 2018
• End date: January 31, 2021
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Agreement: FINEP - PIPE/PAPPE Grant
• Principal Investigator: Amanda Bernardes Muniz
• Grantee: Amanda Bernardes Muniz
• Company: Cellco Biotec do Brasil Ltda
• CNAE: Pesquisa e desenvolvimento experimental em ciências físicas e naturais
• City: São Carlos
• Associated researchers: Larissa Consani Textor ; Maria Amélia Villela Oliva Dotta ; Naiara Utimura Torres
• Associated research grant: 16/00863-7 - Development of DNA polymerases production process with high quality and processivity, AP.PIPE
• Associated scholarship(s): 19/00486-7 - Development of DNA polymerases production process with high quality and processivity, BP.PIPE

Abstract

DNA polymerases are enzymes involved in DNA synthesis by the addition of deoxyribonucleotides, according with a template. It is an enzyme widely used in the in vitro DNA manipulation, including cloning, sequencing and mutagenesis, among other techniques. More specifically, a DNA polymerase from Thermus aquaticus (Taq DNA polymerase) is the most used thermostable DNA polymerase, with applications beyond research use, as an example, the increasing employment in genotyping and diagnostic areas. Although it is considered a basic reagent for biotechnology, the Brazilian market is dependent on DNA polymerases importation, resulting in unfavorable delivery terms and prices. The recombinant Taq DNA polymerase, produced in E. coli, can be obtained by simplified protocols and it has similar biochemical characteristics when compared with native Thermus aquaticus protein, in terms of activity, specificity and thermostability. However, recent studies have demonstrated the presence of microbial DNA and PCR inhibitors in most enzyme preparations, even commercial ones. This contamination is a limiting factor of the use of PCR to detect diluted bacterial DNA in environmental or public health samples. In addition to optimizing the production of enzymes, they are often subject to protein engineering projects, aiming to improve their functional characteristics. Among them, the use of fused-protein domains has shown promising results in the development of DNA polymerases with high processivity. Thus, the aim of this project second phase is the development and scheduling of a productive process for native Taq DNA Polymerase and fusioned to Sso7d domais, an optimized construction with higher processivity, for commercial exploitation in the national market. During of PIPE phase I, the native Taq DNA Polymerase was obtained with high yield and purity, with its activity tested and samples qualified as free of microbial contaminants, therefore, suitable for the purposes of project second phase. Additionally, the modified enzyme is already in the production process to have its functional characterization. It is proposed, therefore, to contribute to the process of nationalization of basic reagents production for molecular biology, improving the competitiveness and research development in Brazil on the world scenario. (AU)