Recombinant Escherichia coli Fe/S biogenesis protein NfuA is produced in E. coli and contains the complete protein sequence from amino acids 1 to 191. The construct includes an N-terminal maltose-binding protein (MBP) tag and a C-terminal 6xHis-Avi tag, which appears to improve solubility and enable affinity purification. The protein undergoes biotinylation and shows a purity level above 85% when analyzed by SDS-PAGE, suggesting it should work well for controlled experimental work.
NfuA likely plays a key role in building iron-sulfur (Fe/S) clusters—cofactors that many cellular processes depend on. This protein seems to help assemble and deliver Fe/S clusters to their target proteins, making it important for cellular iron balance and metabolic function. Because of its involvement in these pathways, NfuA has become an interesting focus for researchers studying bacterial physiology and biochemistry.
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.
Escherichia coli Fe/S biogenesis protein NfuA is a bacterial protein that requires precise folding and iron-sulfur cluster binding for its functional activity in Fe/S cluster assembly and transfer. The E. coli expression system is homologous to this bacterial protein, significantly increasing the probability of correct folding. However, the large N-terminal MBP tag (∼40 kDa) may sterically interfere with the protein's functional domains or interaction interfaces, while the C-terminal biotinylation could potentially affect the protein's C-terminal region. While the protein may be correctly folded, the large tags require experimental validation to confirm functional activity.
1. Protein-Protein Interaction Studies Using Biotin-Streptavidin Capture
This application is conditionally suitable but requires validation. The biotin-streptavidin system provides strong binding for capture experiments, but the large MBP tag may sterically hinder physiological protein interactions. If correctly folded and active (verified), the protein can identify genuine Fe/S cluster pathway partners. If misfolded/tag-interfered (unverified), there is a risk of non-specific binding or interaction failure.
2. Antibody Development and Validation
This application is highly suitable as antibody development relies on antigenic sequence recognition. The full-length protein provides comprehensive epitope coverage, though antibodies may primarily recognize the large MBP tag rather than the NfuA domain if not properly screened.
3. Comparative Biochemical Analysis of Fe/S Biogenesis Proteins
This application requires careful interpretation. The large MBP tag significantly alters the protein's hydrodynamic properties, making direct biophysical comparisons with untagged proteins challenging. If correctly folded (verified), limited comparisons are possible, but tag effects must be considered. The MBP tag's size dominates biophysical properties, complicating direct comparisons with native proteins.
4. ELISA-Based Detection and Quantification Assays
The multiple tags provide excellent flexibility for assay development regardless of native folding. This application is highly suitable due to the versatile tag system. The biotin tag enables efficient streptavidin plate coating, while the His tag provides an alternative capture method. The MBP tag offers additional epitopes for detection antibodies.
Final Recommendation & Action Plan
The homologous E. coli expression system provides favorable conditions for this bacterial Fe/S cluster protein, but the large MBP tag and biotinylation require experimental validation before reliable use in functional interaction studies. Begin with biochemical characterization to assess folding quality through size-exclusion chromatography and validate Fe/S cluster binding activity. Applications 2 and 4 (antibody development and ELISA) can proceed immediately due to their reliance on tag functionality rather than native protein structure. For Application 1, first validate that the tags do not interfere with physiological interactions by testing known binding partners. For Application 3, interpret comparative data with caution due to the MBP tag's dominant effect on biophysical properties. Consider tag removal or using minimal-tag constructs for critical functional studies requiring authentic protein behavior.
