Recombinant Human Apoptosis-associated speck-like protein containing a CARD (PYCARD) is produced in E. coli with an N-terminal 6xHis tag for easier purification. This protein represents amino acids 1-93, making it a partial length construct. Based on SDS-PAGE verification, it appears to be purified to greater than 90% purity, which should ensure high quality for research applications.
PYCARD, also known as ASC, serves as a key adaptor protein in inflammasome formation. These multiprotein complexes seem to play a central role in innate immune responses. The protein is likely critical for activating inflammatory caspases like caspase-1, which then processes and releases pro-inflammatory cytokines. This makes PYCARD an important target for inflammation and immune regulation research.
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.
Based on the provided information, the recombinant human PYCARD is expressed in E. coli, a prokaryotic system that is generally suitable for producing small, soluble protein domains like the PYD domain of PYCARD. PYCARD contains a PYD domain (approximately 1-93aa) that mediates protein-protein interactions in inflammasome assembly. The protein is expressed as a partial fragment (1-93aa) representing the complete PYD domain with an N-terminal 6xHis tag and >90% purity. As a single-domain protein without complex post-translational modifications, it has a reasonable probability of proper folding in E. coli. However, since activity is explicitly unverified and death-fold domains like PYD require precise folding for specific interactions, the protein cannot be assumed to be correctly folded or bioactive without experimental validation of its ability to form functional inflammasome complexes.
1. Protein-Protein Interaction Studies via His-Tag Pull-Down Assays
The N-terminal 6xHis tag enables technical feasibility for pull-down assays. However, if the PYD domain is misfolded, it will not interact physiologically with true binding partners (e.g., ASC, NLRP3). Death-fold domains require precise conformation for specific homotypic interactions. This application should only be pursued after confirming proper folding through biophysical characterization and functional validation.
2. Antibody Development and Validation
The recombinant PYCARD fragment can serve as an effective immunogen for generating antibodies that recognize linear epitopes within the PYD domain. The high purity supports immunization protocols. Validation against full-length PYCARD from mammalian expression systems is recommended to ensure physiological relevance.
3. Biochemical Characterization and Stability Studies
This application is well-suited for initial assessment. Techniques like circular dichroism spectroscopy, size-exclusion chromatography, and thermal shift assays can evaluate the protein's folding state and stability. These studies are valuable for quality control even before functional validation.
4. In Vitro Binding Assays Using ELISA-Based Methods
This application requires proper folding validation. If the PYD domain is misfolded, binding assays will not reflect biological specificity. Death-fold domain interactions require precise three-dimensional conformation. This application should only follow confirmation of proper folding and validation with known binding partners.
Final Recommendation & Action Plan
Given that PYCARD's PYD domain is a relatively small, soluble protein domain that can often fold correctly in E. coli, we recommend first performing validation studies: 1) Biophysical characterization (circular dichroism for characteristic death-fold domain secondary structure, analytical size-exclusion chromatography for oligomeric state); 2) Functional validation using known binding partners (e.g., NLRP3 PYD domain) in pull-down or SPR assays; 3) If possible, comparison with PYD domain from mammalian expression systems. Antibody development can proceed immediately. Interaction studies should await proper folding confirmation. For reliable PYCARD research, include appropriate controls such as known binding partners and validate findings with full-length protein when possible.
