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.
InlA's activity depends on precise tertiary structure, including leucine-rich repeat (LRR) domains and disulfide bond formation. The E. coli system lacks eukaryotic chaperones and may not support the formation of native disulfide bonds or complex folding, often leading to misfolding, aggregation, or inclusion body formation. The S192N and Y369S mutations are located within key functional regions of InlA (e.g., LRR domains), which could further disrupt protein stability, receptor-binding interfaces, or conformational epitopes. The dual tags, particularly the large N-terminal His-tag, may sterically interfere with the N-terminal folding of InlA, which is critical for E-cadherin binding. While it is theoretically possible for the protein to be functional, the absence of validation data and the inherent limitations of E. coli expression mean it cannot be assumed to be correctly folded or bioactive. Experimental verification is essential before any functional application.
1. Protein-Protein Interaction Studies with Human E-cadherin
This recombinant InlA variant could be used to study interactions with human E-cadherin only if its folding and bioactivity are experimentally verified. The dual tags facilitate technical aspects like immobilization for pull-down or SPR assays. However, if the protein is misfolded, its binding interfaces may be altered, leading to inaccurate kinetics (e.g., false negatives in affinity measurements) or non-physiological interactions. The S192N and Y369S mutations might specifically perturb E-cadherin binding, as these residues could lie within critical LRR domains involved in receptor engagement. Any comparative studies with wild-type InlA require prior validation of the mutant’s folding and binding competence.
2. Antibody Development and Immunoassay Applications
The recombinant protein is suitable as an immunogen for generating antibodies targeting linear epitopes, as antibody production often relies on amino acid sequences rather than native conformation. The high purity (>85%) reduces contamination risks. However, if the protein is misfolded, antibodies may not recognize conformational epitopes of native InlA in its physiological context (e.g., on bacterial surfaces or in infection models), limiting utility in neutralization or immunofluorescence assays. The mutations (S192N, Y369S) might generate antibodies with unique specificity, but these could fail to bind wild-type InlA if the mutations alter key epitopes. The tags may also induce tag-specific antibodies, requiring careful screening.
3. Biochemical Characterization and Structural Studies
The protein’s suitability for biochemical or structural analysis (e.g., circular dichroism, thermal stability assays) is highly dependent on correct folding. If misfolded, data on stability or aggregation would not reflect native InlA properties. The mutations might inherently alter folding kinetics or stability, but without validation, conclusions about mutation effects would be unreliable. Techniques like X-ray crystallography would require a homogeneous, correctly folded protein; the tags and potential misfolding could impede crystallization or yield non-native structural insights.
4. Cell Adhesion and Invasion Assays
This application is critically dependent on confirmed bioactivity. InlA’s role in cell adhesion requires precise folding to bind E-cadherin and trigger invasion. If the recombinant protein is misfolded or inactive, coating experiments or bead-based assays would fail to mimic bacterial adhesion, leading to false-negative results. The mutations S192N and Y369S might directly impair E-cadherin binding based on their locations in InlA’s receptor-binding domain. Any cell-based data requires validation using native InlA or complementary infection models.
5. Competitive Binding and Inhibition Studies
This application is fully contingent on functional folding. Competitive binding assays rely on the protein’s ability to interact with E-cadherin or inhibitors in a native-like manner. Misfolding could lead to false negatives (inability to compete) or false positives (non-specific inhibition). The mutations might alter inhibitor sensitivity, but without activity confirmation, screening for blockers would be ineffective. The tags could also sterically hinder inhibitor binding, complicating interpretation.
Final Recommendation & Action Plan
To ensure reliable outcomes, prioritize experimental validation of the protein’s folding and bioactivity before any functional application. Begin with biophysical characterization: use size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to assess oligomeric state and monodispersity, and circular dichroism spectroscopy to compare secondary structure to known spectra of wild-type InlA. Then, perform functional validation: conduct an E-cadherin binding assay (e.g., surface plasmon resonance or ELISA) using a validated E-cadherin construct to confirm binding affinity and specificity. If validation succeeds, the protein can be cautiously used for proposed applications, with disclosures about tag and mutation limitations. If validation fails, restrict use to non-conformation-dependent applications (e.g., linear-epitope antibody production).
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