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Published on: December 30, 2016
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.
Abstract:
The mecA-27R gene, which encodes PBP2a from methicillin-resistant Staphylococcus aureus strain 27R, was modified to remove the putative N-terminal membrane-spanning region, cloned into the T7 RNA polymerase expression vector pET11d, and used to transform Escherichia coli strain BL21(DE3). The majority of PBP2a was expressed in the form of inclusion bodies, which were extracted, denatured, and refolded. The protein was then purified by anion-exchange and size-exclusion chromatography. A 6-liter culture of induced E. coli provided 37 mg of purified PBP2a which was greater than 99% pure. Binding affinities for [3H]benzylpenicillin, imipenem, and L-695,256 (a beta-lactam with high affinity for PBP2a) were shown to be comparable to PBP2a found in membrane preparations of S. aureus strain 27R. A direct binding assay, using 14C-labeled L-695,256 was developed and used to show stoichiometric binding to the refolded, soluble PBP2a. In addition, electrospray mass spectrometry showed that 100% of the refolded PBP2a was covalently bound to the beta-lactam in a stoichiometric fashion. Finally, two mutations of the putative active-site serine showed the predicted loss of covalent binding of the beta-lactam to the PBP2a, demonstrating the high specificity of the soluble binding assay.
Insights
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.
