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High-yield expression, refolding, and purification of penicillin-binding protein 2a from methicillin-resistant
L J Frank1, D Wisniewski, G G Hammond
1Department of Biophysical Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065-0900, USA.
Protein Expression and Purification
|October 1, 1995
Summary
Researchers successfully purified and characterized soluble penicillin-binding protein 2a (PBP2a) from methicillin-resistant Staphylococcus aureus. This breakthrough enables detailed studies of antibiotic resistance mechanisms.
Area of Science:
- Microbiology and Molecular Biology
- Biochemistry and Protein Chemistry
- Antimicrobial Resistance Research
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant public health threat due to its resistance to beta-lactam antibiotics.
- Penicillin-binding protein 2a (PBP2a), encoded by the mecA gene, is a key determinant of MRSA's resistance.
- Studying PBP2a's structure and function is crucial for developing new anti-MRSA strategies.
Purpose of the Study:
- To produce a soluble, highly purified form of PBP2a from MRSA strain 27R for detailed biochemical analysis.
- To characterize the binding affinities of PBP2a to various beta-lactam antibiotics.
- To validate a new direct binding assay for PBP2a-beta-lactam interactions.
Main Methods:
- Modification of the mecA-27R gene to remove the N-terminal membrane-spanning region.
- Expression of recombinant PBP2a in Escherichia coli and purification using anion-exchange and size-exclusion chromatography.
- Binding assays using radiolabeled antibiotics ([3H]benzylpenicillin, imipenem, L-695,256) and electrospray mass spectrometry.
Main Results:
- Obtained 37 mg of >99% pure soluble PBP2a from a 6-liter E. coli culture.
- Purified PBP2a exhibited comparable binding affinities to beta-lactams as native membrane-bound PBP2a.
- Developed a direct binding assay demonstrating stoichiometric and covalent binding of L-695,256 to refolded PBP2a, confirmed by mass spectrometry and site-directed mutagenesis.
Conclusions:
- Successfully produced a functional, soluble PBP2a, overcoming previous challenges with membrane protein purification.
- The developed binding assay is specific and reliable for studying PBP2a-beta-lactam interactions.
- This work provides a foundation for further structural and mechanistic studies of PBP2a and the development of novel antibiotics targeting MRSA.
