Construction of world's first Mycobacterium tuberculosis cytochrome P450 monooxygenase library

Abstract

The actinomycete Mycobacterium tuberculosis causes Tuberculosis (TB), a chronic lung disease in humans and continues to be one of the greatest threats to mankind. Large number of studies showed that M. tuberculosis cytochrome P450 monooxygenases (P450s) can be used as novel drug target. P450s are mixed function oxidoreductases well known for their role in essential cellular anabolic and catabolic processes. Despite the greater importance of M. tuberculosis P450s as novel drug targets, only four M. tuberculosis P450s (apart from highly conserved CYP51) have been functionally characterized for their in vivo role. The major challenges in M. tuberculosis P450 research is expression of M. tuberculosis P450s and identification of substrate(s). This study is aimed to develop world’s first M. tuberculosis P450 expression library by cloning remaining 14 M. tuberculosis P450s. In order to clone 14 M. tuberculosis P450s a cloning strategy was developed such that all 14 M. tuberculosis P450s was cloned into expression vector. In this study, expression vector pINK_A was modified in its multiple cloning site by incorporating more restriction enzyme sites to accommodate all 14 M. tuberculosis P450s. The modified vector was named as pINK_d. Restriction profiling of 14 M. tuberculosis P450s were carried out and suitable restriction enzymes were selected for directional cloning of M. tuberculosis P450s into pINK_d vector. The pINK_d and 14 M. tuberculosis P450 cDNAs were synthesized and all synthesized P450 cDNA were subsequently cloned into pINK_d. The plasmid DNA clones (or constructs) containing M. tuberculosis P450 cDNA was transformed into E. coli DH5α cells and the recombinant E. coli cells were selected on Luria-Bertani agar plates containing ampicillin antibiotic. Plasmids from the recombinant cells were isolated and subjected to restriction enzyme digestion analysis. Restriction enzyme digestion analysis of plasmids revealed that all 14 M. tuberculosis P450s were successfully cloned as correct size of cDNA corresponding to respective M. tuberculosis P450s was released upon digestion with restriction enzymes. This study will pave the way for expression and characterization of M. tuberculosis P450s. Thus developing M. tuberculosis P450 based novel anti-TB drugs. The M. tuberculosis P450 E. coli library developed in this study will be patented after confirming M. tuberculosis P450s expression. Apart from my Masters study, I also supervised two B. Tech student projects and managed to publish an article with students. Furthermore, I also worked on a few other bioinformatics projects and earned co-authorship. Most of my research articles are published in high impact factor journals. The following is a list of my research articles: 1. IKR Kgosiemang (co-author) (2016) Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s. Scientific Reports 6, Article number: 33099. 2. IKR Kgosiemang (co-author) (2015). Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes. Scientific Reports 5, Article number: 11572. 3. NT Mthakathi, IKR Kgosiemang et al, (2015). Cytochrome P450 monooxygenase analysis in free-living and symbiotic microalgae Coccomyxa sp. C-169 and Chlorella sp. NC64A. Alage 30(3):233-239. In addition to the above credits, I was featured on national TV and in newspapers for discovering a novel drug target. I also presented work at both national and international (Canada) conferences.