Prokaryotic expression and purification of transpeptidase domain of penicillin binding protein 2a of methicillin-resistant Staphylococcus aureus

Ji Li, Hui Yang, Rui Hua Tang, Jing Li, Qi Li, Yan Shao, Ting Huyan, Qing Sheng Huang, Jun Ling Shi

Research output: Contribution to journalArticlepeer-review

Abstract

Objective: To express the transpeptidase domain of penicillin binding protein 2a (PI5P2a) of methicillin-resistant Staphylococcus aureus (MRSA) in prokaryotic cells, and purify and identify the expressed product. Methods: The mecAf gene encoding the transpeptidase domain of PBP2a was amplified by PCR and cloned into prokaryotic expression vector pGEX-6p-l. The constructed recombinant plasmid pGEX-6p-meeAf was identified by restriction analysis (BamH I and EcoR I) and sequencing, then transformed to E. coli RL21 and induced with IPTG. The expressed product was purified by affinity chromatography, and identified by SDS-PAGE and Western blot. Results: Both restriction analysis and sequencing proved that recombinant plasmid pGEX-6p-mecAf was constructed correctly. The expressed product, with a relative molecular mass of about 63 000, contained about 14. 5% of total somatic protein and reached a purity of 90% after purification. Conclusion: The prokaryotic expression vector for transpeptidase domain of PBP2a was constructed correctly, and target protein was highly expressed in E. coli, which laid a foundation of preparation of monoclonal antibody and development of rapid detection method for MRSA.

Original languageEnglish
Pages (from-to)497-500
Number of pages4
JournalChinese Journal of Biologicals
Volume28
Issue number5
StatePublished - 20 May 2015

Keywords

  • Gene expression
  • Methicillin-resistant Staphylococcus aureus (MRSA)
  • Penicillin binding protein 2a (PBP2a)
  • Prokaryotic cells
  • Transpeptidase domain

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