Novel Antimicrobial Agents Targeting Flap Endonuclease

Abstract

Streptococcus pneumoniae (Sp) is considered a major global health concern causing a wide range of diseases mainly to vulnerable populations. As the threat of antimicrobial resistance is increasing, the need to investigate new therapeutic targets are pivotal. Bacterial flap endonuclease (FEN) domains of DNA polymerase I enzymes are structure-specific nucleases that are essential in DNA replication and repair as they are responsible for cleaving 5′ flap structures arising during Okazaki fragment processing. This project aimed to assess SpFEN domain protein-DNA interactions. Two pUC57-Kan vectors containing the synthetic SpFEN genes, wildtype (active) and mutant (inactive), were cloned into a suitable expression vector pJONEX4 using EcoRI and HindIII restriction enzymes and transformed into competent M72 Escherichia coli cells. The expression of recombinant protein was optimised to maximise the soluble yield and produce functional SpFEN protein. The harvested and lysed cells, containing the wildtype vector, were then subjected to ion exchange chromatography and affinity chromatography using the AKTA pure purification system followed by size exclusion chromatography to ensure purity of target protein and homogeneity. Chromatographic analysis was displayed on software where it indicated a highly pure preparation of target protein and confirmed by SDS-PAGE. The nuclease activity of recombinant SpFEN wildtype was then evaluated by conducting zymogram to detect enzyme cleavage efficiency. A robust endonucleolytic activity of enzyme was exhibited compared to the mutant SpFEN type where no degradation of DNA substrate was observed indicating lack of enzymatic activity; which was consistent with previous studies on a bacteriophage FEN (Feng et al., 2004). To identify inhibitors of SpFEN activity, a Forster Resonance Energy Transfer (FRET) activity assay was performed to screen a collection of chemical compounds. Together, such data contribute to understanding the promising antimicrobial target FEN interactions which is essential for the development of new, safe and effective therapeutic agents.