Zuma, LKGasa, NLMazibuko, XSimelane, BCPillay, PriyenKwezi, LusisizweTsekoa, Tsepo LPooe, OJ2022-11-212022-11-212022-04Zuma, L., Gasa, N., Mazibuko, X., Simelane, B., Pillay, P., Kwezi, L., Tsekoa, T.L. & Pooe, O. et al. 2022. Recombinant expression, purification and PEGylation of DNA Ligases. <i>Protein and Peptide Letters.</i> http://hdl.handle.net/10204/125251875-53050929-8665Doi: 10.2174/0929866529666220426122432http://hdl.handle.net/10204/12525Background: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are routinely used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. Objective: The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases. Method: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins were conjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected various PEGylation conditions. Results: In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-fold for plasmid ligations and reduced the formation of protein aggregates. Conclusion: Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.AbstractenDNA ligasesPEG conjugationProtein expression and purificationProtein PEGylationSite-specific PEGylationArticleZuma, L., Gasa, N., Mazibuko, X., Simelane, B., Pillay, P., Kwezi, L., ... Pooe, O. (2022). Recombinant expression, purification and PEGylation of DNA Ligases. <i>Protein and Peptide Letters</i>, http://hdl.handle.net/10204/12525Zuma, LK, NL Gasa, X Mazibuko, BC Simelane, Priyen Pillay, Lusisizwe Kwezi, Tsepo L Tsekoa, and OJ Pooe "Recombinant expression, purification and PEGylation of DNA Ligases." <i>Protein and Peptide Letters</i> (2022) http://hdl.handle.net/10204/12525Zuma L, Gasa N, Mazibuko X, Simelane B, Pillay P, Kwezi L, et al. Recombinant expression, purification and PEGylation of DNA Ligases. Protein and Peptide Letters. 2022; http://hdl.handle.net/10204/12525.TY - Article AU - Zuma, LK AU - Gasa, NL AU - Mazibuko, X AU - Simelane, BC AU - Pillay, Priyen AU - Kwezi, Lusisizwe AU - Tsekoa, Tsepo L AU - Pooe, OJ AB - Background: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are routinely used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. Objective: The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases. Method: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins were conjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected various PEGylation conditions. Results: In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-fold for plasmid ligations and reduced the formation of protein aggregates. Conclusion: Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases. DA - 2022-04 DB - ResearchSpace DP - CSIR J1 - Protein and Peptide Letters KW - DNA ligases KW - PEG conjugation KW - Protein expression and purification KW - Protein PEGylation KW - Site-specific PEGylation LK - https://researchspace.csir.co.za PY - 2022 SM - 1875-5305 SM - 0929-8665 T1 - Recombinant expression, purification and PEGylation of DNA Ligases TI - Recombinant expression, purification and PEGylation of DNA Ligases UR - http://hdl.handle.net/10204/12525 ER -25914