Development of DNA polymerases production process with high quality and processivity

• Grant number: 16/00863-7
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: October 01, 2016
• End date: October 31, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Amanda Bernardes Muniz
• Grantee: Amanda Bernardes Muniz
• Company: Cellco Biotec do Brasil Ltda
• Associated researchers: Maria Amélia Villela Oliva Dotta
• Associated research grant(s): 17/12334-1 - Development of DNA polymerases production process with high quality and processivity, AP.PIPE
• Associated scholarship(s): 16/20162-3 - Development of DNA polymerases production process with high quality and processivity, BP.PIPE

Abstract

DNA polymerases are ubiquitous enzymes that synthesizes complementary DNA strand by adding deoxyribonucleotides to a double-stranded DNA primer, in accordance with a template. It is an enzyme widely used in the in vitro DNA manipulation, including cloning, sequencing, and mutagenesis, among others techniques. The DNA polymerase from Thermus aquaticus (Taq polymerase) is the most used thermostable DNA polymerase. It is now used not only in research, but also in the general human society, including genotyping and diagnosis. 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. Recombinant Taq DNA polymerase produced in E. coli has similar biochemical characteristics when compared with native Thermus aquaticus protein as for its activity, specificity and thermostability. The production of DNA Taq recombinant polymerase is therefore possible and simple protocols can be applied, especially by exploiting its thermostable nature. However, recent studies have demonstrated the presence of microbial DNA in several commercial preparations of DNA polymerases. This contamination is a limiting factor of the use of PCR to detect diluted bacterial DNA in environmental or public health samples. Additionally, increasing the performance of DNA polymerases is often subject to protein engineering projects. 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 is the development of a productive process for Taq DNA polymerase with high purity level, yield as well as reduced microbial DNA contamination. In addition, it shall be obtained a novel DNA polymerase enzyme, derived from the fusion of the Taq polymerase with the DNA binding domain Sso7d, resulting in a higher processivity and, consequently, reducing the required time for routine PCR reactions. The steps involved in this process consist in: i. cloning the fused DNA construct of Thermus aquaticus polymerase and Sso7d from Sulfolobus solfataricus; ii. establishing a bacterial expression system for heterologous protein production; iii. standardization of protocols for Taq and Sso7d-Taq production and purification; iv. characterization of the produced enzymes activities compared to other available polymerases. The expected result at this stage is to obtain the Taq polymerase enzyme and also its hybridized version with higher processivity and both free of microbial DNA contamination. The feasibility of this production process and the new product will match the market-production scale and reach the national market with higher quality enzymes and more attractive prices, besides the development of newer technologies in Brazil. 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)