Recombinant Trichosanthes kirilowii Ribosome-inactivating protein alpha-trichosanthin is produced using an E. coli expression system and carries an N-terminal 10xHis tag. This protein contains the complete mature sequence from amino acids 24-270. SDS-PAGE analysis indicates the product reaches greater than 90% purity, which appears to meet quality standards for research use.
Alpha-trichosanthin comes from Trichosanthes kirilowii and is recognized for its capacity to shut down protein synthesis through ribosome inactivation. The mechanism involves removing a specific adenine base from ribosomal RNA—a modification that seems critical for its research applications in protein synthesis regulation. Scientists often turn to this protein when studying ribosome structure and function, though the complexity of these systems means results may vary.
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.
Trichosanthes kirilowii alpha-trichosanthin is a ribosome-inactivating protein that requires precise folding, proper disulfide bond formation (with multiple conserved disulfide bonds), and specific active site conformation for its N-glycosidase activity. The E. coli expression system cannot provide the eukaryotic oxidative environment for correct disulfide bond formation. The N-terminal 10xHis-tag may sterically interfere with the protein's functional domains. While the mature protein region (24-270aa) contains the catalytic domain, the probability of correct folding with functional ribosome-inactivating activity requires experimental validation of disulfide bond formation and enzymatic activity.
1. Ribosome-Inactivating Protein Biochemical Characterization
This application carries a significant risk without functional validation. Trichosanthin's enzymatic activity requires precise disulfide bond formation and proper active site conformation. If correctly folded and active (verified through rRNA depurination assays), the protein may be suitable for kinetic studies. If misfolded/inactive (unverified), enzymatic measurements will yield biologically meaningless results.
2. Protein-Protein Interaction Studies
This application requires proper folding validation. Trichosanthin interactions with ribosomal proteins require precise tertiary structure and correct disulfide bonding. If correctly folded (verified), the protein may identify physiological interaction partners. If misfolded/unverified, there is a high risk of non-specific binding or failure to replicate genuine ribosomal interactions.
3. Antibody Development and Validation
This application is highly suitable as antibody development relies on antigenic sequence recognition rather than functional enzymatic activity. The full-length mature protein provides comprehensive epitope coverage for generating trichosanthin-specific antibodies. The high purity (>90%) ensures minimal contamination-related issues during immunization protocols.
4. Structural and Biophysical Analysis
These studies are essential for determining folding status. Techniques should include circular dichroism spectroscopy to assess secondary structure, size-exclusion chromatography to evaluate oligomeric state, and disulfide bond analysis. However, the His-tag may interfere with high-resolution structural studies and functional domain accessibility.
Final Recommendation & Action Plan
The E. coli-expressed trichosanthin with His-tag may not be properly folded for functional applications due to the lack of eukaryotic disulfide bond formation machinery. Begin with Application 4 (Structural Characterization) to assess folding quality through CD spectroscopy, SEC, and validate enzymatic activity using rRNA depurination assays before considering functional applications. Applications 1 and 2 require rigorous functional validation. Application 3 (antibody development) can proceed immediately. For reliable trichosanthin research requiring native functionality, use eukaryotic expression systems that support proper disulfide bond formation, or implement refolding protocols with extensive activity validation.
