Recombinant Expression, Purification and PEGylation of DNA Ligases

Abstract

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.

Research progress on the PEGylation of therapeutic proteins and peptides (TPPs)

“…Other groups on TPPs can also be utilized as potential sites for PEGylation, such as amino acid residues located at the protein or peptide's C-terminus (Cazalis et al, 2004), glycosylation sites consisting of serine and threonine residues (Giorgi et al, 2014), and histidine residue (Wylie et al, 2001;Zuma et al, 2022b;Zuma et al, 2022a;Khataminezhad et al, 2023). Both IFN α-2b and IFN β-1b were fused to the Mycobacterium xenopi GyrA intein and expressed in Escherichia coli.…”

Protein PEGylation: Navigating Recombinant Protein Stability, Aggregation, and Bioactivity

“…Residue specific PEGylation was shown to strengthen the Glu12-Arg14 salt-bridge by shielding it from the interference of water molecules [ 22 ]. Recently, Zuma and colleagues (2022) recently demonstrated that site-specific PEGylation enhances the biological activity and stability of recombinant DNA ligase proteins [ 23 ]. Cooper and colleagues showed that proteins treated with mono-sulfone-PEG retained higher significantly conjugation [ 24 ].…”