Recombinant Citrus unshiu Limonoid UDP-glucosyltransferase is produced through a baculovirus expression system, which appears to deliver solid fidelity and consistency. The complete protein covers amino acids 1-511 and includes an N-terminal 10xHis tag plus a C-terminal Myc tag - both designed to make purification and detection more straightforward. SDS-PAGE analysis indicates the product reaches purity levels above 85%, suggesting it should work well for different research applications.
Limonoid UDP-glucosyltransferase is an enzyme that handles the glucosylation of limonoids, which are secondary metabolites commonly found in citrus plants. This enzyme may play a significant role in modifying these limonoids, likely affecting both their solubility and bioactivity. It represents an important target for studies examining plant biochemistry and efforts to improve citrus fruit quality.
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. In Vitro Enzyme Activity Characterization
This recombinant limonoid UDP-glucosyltransferase could help establish and refine enzymatic assays for studying how limonoid compounds undergo glycosylation. Scientists might explore the enzyme's substrate specificity by testing different limonoid substrates and UDP-sugar donors under controlled laboratory conditions. The His and Myc tags should make protein purification and detection relatively straightforward, allowing for accurate enzyme concentration measurements during kinetic studies. Such work would likely generate essential biochemical data on this citrus-specific glycosyltransferase family.
2. Substrate Specificity and Kinetic Studies
The purified enzyme can be used to systematically examine its catalytic behavior with various limonoid substrates typically present in citrus species. Scientists can measure Km and Vmax values for different substrate combinations to better understand the enzyme's efficiency and preferences. The high purity level (>85%) should help ensure reliable kinetic measurements without interference from other proteins. These studies may contribute valuable insights into how limonoid metabolism actually works in citrus plants.
3. Antibody Development and Immunoassay Applications
The dual-tagged recombinant protein appears well-suited as an immunogen for creating specific antibodies against citrus limonoid UDP-glucosyltransferase. Both tags - the N-terminal His tag and C-terminal Myc tag - should simplify purification and detection during antibody screening. Any antibodies developed this way could prove useful in Western blotting, immunoprecipitation, or ELISA-based detection systems when studying this enzyme in citrus tissue extracts. Such immunological tools might help researchers track the enzyme's expression patterns and determine where it localizes within cells.
4. Protein-Protein Interaction Studies
The tagged recombinant protein could work well in pull-down assays aimed at finding potential protein partners that interact with limonoid UDP-glucosyltransferase during citrus metabolism. The His tag allows for attachment to metal affinity matrices, while the Myc tag provides a way to detect and confirm the bait protein's presence. Scientists can expose the immobilized enzyme to citrus protein extracts to capture and identify interacting proteins using mass spectrometry analysis. This approach might reveal regulatory mechanisms or metabolic complexes that involve limonoid glycosylation.
5. Comparative Enzyme Studies Across Citrus Species
This Citrus unshiu enzyme could serve as a reference point for comparative studies with similar enzymes from other citrus species or varieties. Researchers might compare catalytic properties, substrate preferences, and responses to inhibitors between different citrus limonoid UDP-glucosyltransferases. The standardized expression system and purification tags should help ensure consistent protein quality for reliable comparative work. Studies like these may provide insights into how limonoid metabolism evolved and diversified across different citrus species.
