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.
1. Ribosome-Inactivating Protein Biochemical Characterization
Researchers can use this recombinant alpha-trichosanthin to examine the enzymatic properties and substrate preferences of ribosome-inactivating proteins (RIPs). In vitro ribosome binding and inactivation assays offer one approach. The N-terminal 10xHis tag makes protein purification and immobilization more straightforward for kinetic studies with rabbit reticulocyte lysate or wheat germ translation systems. Scientists might investigate the protein's N-glycosidase activity on ribosomal RNA, though determining optimal conditions like pH, temperature, and ionic strength often requires careful optimization.
2. Protein-Protein Interaction Studies
The His-tagged recombinant protein works well for pull-down assays designed to identify cellular binding partners and ribosomal components that may interact with alpha-trichosanthin. Nickel-affinity chromatography allows researchers to capture protein complexes from cell lysates, followed by analysis through mass spectrometry or Western blotting. This strategy could help map the protein's cellular targets and clarify how it recognizes and binds ribosomes, though complex cellular environments sometimes complicate interpretation.
3. Antibody Development and Validation
This purified recombinant protein can function as an immunogen for producing polyclonal or monoclonal antibodies against alpha-trichosanthin. The high purity level (>90%) likely reduces cross-reactivity with bacterial contaminants during immunization. Generated antibodies can be tested using ELISA, Western blot, and immunoprecipitation assays with the recombinant protein serving as a positive control. This approach enables detection and quantification of native alpha-trichosanthin in plant extracts, though antibody specificity varies depending on the chosen epitopes.
4. Structural and Biophysical Analysis
The recombinant protein provides adequate material for detailed structural studies using circular dichroism spectroscopy, dynamic light scattering, and analytical ultracentrifugation. Researchers can examine the protein's secondary structure, thermal stability, and oligomerization behavior across different buffer conditions. The His tag allows for oriented attachment to sensor surfaces in surface plasmon resonance experiments, which may help analyze binding kinetics with ribosomal subunits or other large molecular targets. However, the presence of the tag itself could potentially influence some measurements.
