Recombinant Escherichia coli DNA gyrase subunit A (gyrA) (D87G)

Recombinant Escherichia coli DNA gyrase subunit A (gyrA) (D87G) is expressed in E. coli and comes with an N-terminal 10xHis-tag that makes purification straightforward. The protein appears as the full-length mature form, spanning amino acids 2-875 with the D87G mutation. SDS-PAGE analysis indicates purity greater than 85%, which should provide adequate reliability for most research applications.

DNA gyrase subunit A represents a crucial piece of the bacterial DNA gyrase enzyme - the molecular machine that introduces negative supercoils into DNA. This supercoiling process is essential for both DNA replication and transcription to proceed normally. Researchers have shown considerable interest in this protein because of its central role in DNA manipulation and its connection to antibiotic resistance mechanisms. A deeper understanding of how it functions may help guide the development of new antimicrobial agents.

Potential Applications

Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.

E. coli DNA gyrase subunit A (GyrA) is a large, complex bacterial protein that requires proper folding and dimerization for its function in DNA supercoiling. The D87G mutation is a known quinolone resistance mutation that specifically affects drug binding but does not necessarily disrupt the protein's overall folding or ability to form complexes with GyrB. The E. coli expression system is homologous for this bacterial protein, increasing the likelihood of correct folding. The N-terminal His-tag is small and unlikely to significantly interfere with protein function. Therefore, this recombinant GyrA(D87G) has a high probability of being correctly folded and capable of forming functional complexes with GyrB, though its drug resistance profile will differ from wild-type.

1. Protein-Protein Interaction Studies with DNA Gyrase Subunit B

Any interaction data would be biologically irrelevant without validation using a natively folded protein. The homologous expression system and minimal His-tag support native conformation. The D87G mutation primarily affects the drug-binding site rather than the GyrB interaction interface, making it reliable for complex formation studies. Co-immunoprecipitation and pull-down assays should yield physiologically relevant results.

2. Antibody Development and Validation

This protein serves as an excellent immunogen for generating GyrA-specific antibodies. The full-length sequence ensures comprehensive coverage of the epitope. While the D87G mutation may create a unique epitope, most antibodies will recognize both wild-type and mutant GyrA. The high purity supports reliable antibody production and validation.

3. Drug-Protein Interaction Screening

This application is highly suitable for studying quinolone resistance mechanisms. The D87G mutation is known to reduce quinolone binding affinity, making this protein ideal for comparative studies with wild-type GyrA. Binding assays can quantitatively characterize the resistance profile and screen for alternative inhibitors that may overcome this resistance.

4. Structural and Biophysical Characterization

This is a priority application for understanding the structural basis of quinolone resistance. Biophysical techniques can reveal how the D87G mutation affects protein conformation, stability, and oligomerization without disrupting overall folding. Comparative studies with wild-type GyrA will provide insights into resistance mechanisms.

Final Recommendation & Action Plan

This recombinant GyrA(D87G) is highly suitable for all proposed applications due to its homologous expression system and the specific nature of the D87G mutation that primarily affects drug binding rather than protein folding or partner interactions. The recommended approach is to begin with Application 4 (Structural and Biophysical Characterization) to confirm proper folding and understand the mutation's structural consequences. Then proceed to Application 3 (Drug-Protein Interaction Screening) to characterize the quinolone resistance profile. Applications 1 and 2 (Interaction Studies and Antibody Development) can be conducted in parallel with high confidence. This protein represents a particularly valuable tool for studying antibiotic resistance mechanisms while maintaining utility for general gyrase research applications. All studies should include appropriate wild-type controls when investigating mutation-specific effects.