The Identification Of A New Pdf Gene From Enterococcus Faecium And Establishing Of A New Screening Model Targeted On PDF For Novel Broad Spectrum Antibiotics

Vancomycin was once considered a drug of the last resort for treating resistant Gram-positive bacterial infections, and with emergence of vancomycin-resistance Enterococci (VRE), the last resort is faced with a serious crisis, therefore, searching for new antibiotics with novel mechanisms and potent activity against VRE has become an urgent need. It is common knowledge that exploring the new molecular targets and establishing the new screening models based on the targets is the important key of discovering new dgugs. Peptide Deformylase (PDF) catalyzes the removal of the N-terminal formyl group from nascent polypeptide in eubacteria and is an essential enzyme for growth and multiplication of bacteria but is absent from mammalian cells, therefore it is regarded as one of most promising targets for the discovery of new antibiotics with broad anti-bacteria spectrum.Till now more than 90 pdf DNA sequences from various bacteria have been identified, but few of them have been so far clarified thoroughly on enzymatic properties.In this study, adopting the method of bioinformatics, the orthologous of peptide deformylase from E. faecium was identified, the amino acid sequences of PDF from E.coli, Str.pneumoniae etc were used in BLAST search at NCBI with E. faecium genome. The homologue sequence of the possible peptide deformylase from E. faecium was amplified by PCR and cloned to the protein expressing vector pET-30a-(+) and was overexpressed in the host cells E.coli B121(DE3). The purified PDF of E.faecium was further identified by Western blotting and its enzyme activity and characterization of the PDF was further studied in respect of the effects of substrate concentrations, pH and temperature on the PDF activity and the kinetics property of PDF.The sequence of the new gene pdf encoding PDF of E.faecium has been recorded into Genebank (Accession AY238515).