Production and characterization of DNA ligases isolated from Kogelberg Biosphere metagenomics library.

Zuma, Lindiwe Khumbuzile.

Production and characterization of DNA ligases isolated from Kogelberg Biosphere metagenomics library.

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Zuma_Lindiwe_Khumbuzile_2021.pdf (3.45 MB)

Date

2021

Authors

Zuma, Lindiwe Khumbuzile.

Abstract

Microbial enzymes have been described as an underutilized source of novel enzymes with potential economic advantages. Recently discovered enzymes such as DNA ligase from metagenomic studies, have been shown to achieve great potential in transforming the reagent market specifically in the African continent. Reagent proteins are frequently utilized in the research field widely and are prone to protein degradation and shelf-life reduction. Hence, this study sought to improve biological activity, shelf life and stability of the two DNA ligases identified from Kogelberg Biosphere metagenomics library. Two recombinant DNA ligases expression studies were done using E.coli BL21 and purification studies were done subsequently using affinity chromatography. Both recombinant DNA ligases (Ligsv081 & LigpET30) were successfully expressed and purified as homogenous proteins. In this study two approaches were used to enhance the biological DNA ligases, the first approach used was PEGylation. The purified proteins were conjugated to PEG using site-specific PEGylation and non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze the secondary structure of the PEG conjugated DNA ligases. Thermal stability assays were then employed to assess protein stability in the conjugation with PEG. Site-specific PEGylation enhanced ligase activity and reduced the formation of protein aggregates. The second approach involved DNA ligase co-expression in the presence of PfHsp70 or chimeric transcription factor, respectively. Protein co-expression and co-purification assays were conducted. The co-expression and co-purification assays of both proteins with chimeric transcription factor (cTF) were successful, followed by co-expression and co-purification of LigpET30-PfHsp70. Ligation assays were conducted to assess bioactivity of proteins. All DNA ligase complexes were functional and their melting point was increased. Taken together, site-specific PEGylation and protein co-expression with PfHsp70 potentially extended the shelf-life and stability of the proteins. PEGylation strategies and co-expression strategies can potentially be used to enhance reagents in diagnostic and therapeutic tools in molecular biology field.