Antarctic recombinant proteins: Cloning and characterization of a recombinant thermolabile uracil DNA glycosylase for its application in One step reactions

BEIRO JULIETA MAILEN; RIPOLL LUCAS; BORIO CRISTINA SILVIA; BILEN MARCOS FABIAN

Ciudad de Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Recombinant protein production continues to be one of the most significant contributions of modern biotechnology. They represent a fundamental part in countless processes, being an indispensable tool inthe scientific, technological and industrial fields. The impact of recombinant proteins can be seen in numerous areas, and this market niche is continuously growing and the possibility of generating new variants is an area of great interest.Uracil DNA glycosylases (UDG) are a highly conserved and specific type of DNA repair enzymes that catalyze the release of uracil molecules in DNA by hydrolyzing the N- glycosidic bond, leaving a basic site.The most popular biotechnological applications are the elimination of carryover contamination in real time PCR reactions and the adapter ligation for some massive sequencing technologies.Although the incorporation of UDG into nucleic acid amplification systems has solved the huge problem of contamination caused by traces of products from previous PCR reactions in a rather simple way, certain drawbacks can also be found in its use. Uracil DNA glycosylases from mesophilic microorganisms, such as E. coli, have the particularity of retaining their activity even up to temperatures close to 85oC. This represents a challenge when working with One Step or isothermal systems, where a UDG that maintains activity at the temperature at which the amplification reaction occurs would imply the degradation of the desired product. Therefore, a thermolabile UDG would solve this problem. As they are inactivated at low temperatures (>42° C), they can be coupled to both One Step RT- qPCR and isothermal systems, without affecting the amplification reaction. The main objective of this project is to obtain a thermolabileuracil DNA glycosylase, which can be coupled to one step RT-PCR and isothermal amplification systems.In order to do this, sequences that could meet the characteristics sought were selected from a gene database through bioinformatic analysis. A sequence from a psychrophilic microorganism isolated in the Argentine Antarctic territory was selected. Subsequently, different genetic modifications necessary to optimize its synthesis, cloning, expression and subsequent purification of the expression product were designed.The purified protein was characterized for its uracil DNA glycosylase activity by real-time PCR. The enzyme showed an improved UDG activity between 10°C and 25°C, in comparison to other commercial enzymes.On the other hand, the thermostability tests showed that the enzyme is completely inactivated at 42oC.This feature would allow the use of this protein in integrated enzyme systems, that allow many stages of a biotechnological process to be carried out at different temperatures in a single reaction tube, reducingthe time per reaction and optimizing the workflow.Obtaining this tool is of high impact in the field of molecular biology, and has great potential as a biotechnological product.