Recombinant Listeria monocytogenes serovar 1/2a Internalin-A (inlA) (S192N,Y369S) is produced in E. coli and comprises a partial sequence from amino acids 32 to 414, incorporating the S192N and Y369S mutations. This protein carries dual tags: an N-terminal 10xHis-tag and a C-terminal Myc-tag, which help with purification and detection. SDS-PAGE analysis shows it's purified to greater than 85% purity, making it appropriate for various research applications.
Internalin-A (inlA) is a surface protein from Listeria monocytogenes that appears crucial for mediating bacterial entry into host cells. The protein plays a significant role in listeriosis pathogenesis by helping the bacteria invade non-phagocytic cells. This makes it a key component when studying bacterial-host interactions. It's particularly valuable for research aimed at understanding how bacterial infections develop and how immune responses work.
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.
1. Protein-Protein Interaction Studies with Human E-cadherin
This recombinant Internalin-A variant can investigate binding interactions with human E-cadherin, which is the known cellular receptor for InlA. The dual His and Myc tags provide flexible detection and purification options for pull-down assays, surface plasmon resonance, or co-immunoprecipitation experiments. The S192N and Y369S mutations may alter binding specificity or affinity compared to wild-type InlA - though the exact effects remain to be determined. This makes the variant valuable for structure-function relationship studies. Researchers can compare binding kinetics between this mutant and wild-type protein to understand what role these specific residues play in host cell recognition.
2. Antibody Development and Immunoassay Applications
The dual-tagged recombinant protein works well as an immunogen for generating antibodies specific to Listeria monocytogenes InlA. While the His-tag helps with protein purification for immunization protocols, the Myc-tag provides an additional epitope for detection in screening assays. This protein proves useful in ELISA development, Western blot standardization, and immunofluorescence assay optimization. The specific mutations present might generate antibodies with unique specificity profiles that could help distinguish between different InlA variants.
3. Biochemical Characterization and Structural Studies
The purified recombinant protein allows for detailed biochemical analysis. This includes protein folding studies, thermal stability assessments, and proteolytic sensitivity mapping. The N-terminal His-tag makes purification straightforward using metal affinity chromatography. This provides sufficient quantities for biophysical characterization techniques such as circular dichroism spectroscopy or dynamic light scattering. Researchers can investigate how the S192N and Y369S mutations affect protein stability, conformation, or aggregation behavior compared to the wild-type sequence - though these effects aren't yet fully characterized.
4. Cell Adhesion and Invasion Assays
Cell-based assays using this recombinant InlA variant can study bacterial adhesion mechanisms with cultured human epithelial cells. The protein can be coated onto surfaces or beads to mimic how bacteria present InlA and assess cellular binding capacity. The Myc-tag allows immunofluorescent tracking of protein localization during cell interaction studies. These experiments may help clarify what functional consequences the specific amino acid substitutions have on host cell recognition and binding efficiency.
5. Competitive Binding and Inhibition Studies
The recombinant protein serves as a useful tool for screening potential InlA-blocking compounds or competitive inhibitors in research settings. Dual tags make quantitative detection possible in competition assays where test compounds are evaluated for their ability to disrupt InlA-E-cadherin interactions. This application is particularly relevant for understanding bacterial pathogenesis mechanisms and identifying molecular targets for basic research. The specific mutations might reveal altered sensitivity to certain inhibitory compounds compared to wild-type InlA, though this requires further investigation.
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