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. 2001 Apr;45(4):1053-7.
doi: 10.1128/AAC.45.4.1053-1057.2001.

Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells

C M Apfel  1 , S Evers, C Hubschwerlen, W Pirson, M G Page, W Keck
Affiliations

Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells

C M Apfel et al. Antimicrob Agents Chemother. 2001 Apr.

Abstract

An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

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Figures

FIG. 1
FIG. 1
Schematic representation of plasmids used in this study as templates in the cell-free or in vivo TC-TL assays. For details, see Materials and Methods. Abbreviations for restriction enzymes: Ba, BamHI; Nc, NcoI. +, yes; −, no.
FIG. 2
FIG. 2
Schematic outline of the procedures for the detection of PDF inhibitor activity. The cell-free and whole-cell assays using detection of radioactivity from a polyacrylamide gel are shown on the left, and the higher-throughput assay using Ni-chelate-coated scintillant plates for detection is shown on the right. Incorporation of 35S- or 3H-labeled methionine, respectively, was used for labeling the N terminus.
FIG. 3
FIG. 3
Autoradiography of SDS-PAGE gels from cell-free and in vivo TC-TL assays. The cell-free (A) or the intact-cell in vivo TC-TL standard (B) assay was performed using pTC/TL as a template in the presence of increasing amounts of potential PDF inhibitors (as indicated). As a positive control, the reaction using pTC/TL-con as a template is also shown. ∗, expected protein for pTC/TL; o, expected protein for pTC/TL-con. A representative experiment is shown.
See this image and copyright information in PMC

References

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