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Molecular and Cellular Biochemistry

, Volume 357, Issue 1–2, pp 47–54 | Cite as

Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus

  • Xuchun Che
  • Jinwei Hu
  • Lijuan Wang
  • Zhifeng Zhu
  • Qiong Xu
  • Junqiang Lv
  • Zheng Fu
  • Yajun Sun
  • Jia Sun
  • Gang Lin
  • Rong Lu
  • Zhi Yao
Article
  • 317 Downloads

Abstract

Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni2+-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl2 to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl3 to the growth medium for E. coli BL21 (DE3) cells and adding FeCl3 and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC50 value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.

Keywords

Peptide deformylase E. coli S. aureus Expression Purification Activity 

Notes

Acknowledgments

This study was supported by Grant sponsor: Ph.D. Programs Foundation of Ministry of Education of China (Grant number: 20101202110001); and Natural Science Foundation of Tianjin (Grant number: 09JCZDJC19700); and Cooperation Project in Industry, Education and Research of Guangdong Province (Grant number: 2009B090300430).

Conflict of interest

None.

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Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Xuchun Che
    • 1
  • Jinwei Hu
    • 1
  • Lijuan Wang
    • 1
  • Zhifeng Zhu
    • 1
  • Qiong Xu
    • 1
  • Junqiang Lv
    • 1
  • Zheng Fu
    • 1
  • Yajun Sun
    • 1
  • Jia Sun
    • 1
  • Gang Lin
    • 2
  • Rong Lu
    • 1
    • 2
  • Zhi Yao
    • 1
    • 3
  1. 1.Department of ImmunologyTianjin Medical UniversityTianjinChina
  2. 2.Shenzhen Kangzhe Pharmaceutical Co., Ltd.ShenzhenChina
  3. 3.Key Laboratory of Immuno Microenvironment and Disease of the Educational Ministry of ChinaTianjinChina

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