Abstract：
Objective To establish and validate a high throughput model for screening of Mycobacterium tuberculosis peptide deformylase (PDF) inhibitors as potential antituberculosis drugs, and to perform preliminary screening with microbial fermentation extracts.
Methods The H37Rv PDF coding gene def was amplified with PCR and cloned into the expression vector pET-28a. The recombinant PDF was over-expressed and purified. A high throughput model was established based on the detection of fluorescence intensity, which is caused by the reaction of fluorescamine and free NH2 released by PDF from substrate for-Met-Ala-Ser. Using the assay, 12 400 microbial fermentation extracts were screened, and positive samples’ antibacterial activity to Mycobacterium smegmatis mc2 155 were tested using K-B method. Finally, cytotoxicity of positive samples was assessed.
Results The expression vector pET-28a-def was constructed. The established model was feasible and stable for drug screening.
12 400 microbial fermentation extracts were screened and 8 positive samples were obtained, showing a 0.06% positive rate. Five of them had relatively low cytotoxicity.
Conclusion In this work, we developed a sensitive and reproducible assay for screening of PDF inhibitors. The positive samples are worth investigating in the future.
2012, Vol. 7 
