세균의 Peptide Deformylase(PDF)를 억제하는 새로운 항균물질의 스크리닝

Screening of New Antibiotics Inhibiting Bacterial Peptide Deformylase (PDF)

Peptide deformylase (PDF) is essential and unique to bacteria, thus making it an attractive target for the discovery of novel antibacterial drugs. PDF deformylates the N-formylmethionine of newly synthesized polypeptides in prokaryotes. In this study, a pdf gene from Staphylococcus aureus 6538p was cloned in pET-14b vector and PDF protein was over-produced in Escherichia coli BL21 (DE3). NH2-terminal His-tagged PDF protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography. Enzymatic activity of purified 6xHis- tagged PDF was tested on the substrate (formyl-Methionine-Alanine-Serine) by formate dehydrogenase-coupled spectrometric assay of peptide deformylase. For the discovery of new PDF inhibitors from chemical libraries and culture broths of soil bacteria, a target-oriented screening sisters using a 96-well plate was developed. About 3,000 commercial chemical libraries were tested in this screening system, and 2 chemicals (0.07%) among them showed an inhibitory activity against PDF enzyme. This result showed that a new screening system can be used for the discovery of new PDF inhibitors.