Recombinant Viola hederacea Root cyclotide 1
Description
Biosynthesis and Tissue-Specific Expression
vhr1 is natively expressed in Viola hederacea roots, distinguishing it from most cyclotides found in aerial plant parts . Its precursor gene encodes a protein processed to yield the mature cyclotide via enzymatic cleavage and cyclization. Unlike Fabaceae cyclotides embedded in albumin precursors (e.g., Cter M ), Violaceae cyclotides like vhr1 derive from precursors with conserved endoplasmic reticulum (ER) signal sequences and prodomains .
Biological Activities and Mechanisms
vhr1 exhibits functional parallels to other cyclotides:
-
Insecticidal activity: Disrupts lipid membranes via pore formation, a mechanism shared with cyclotides like kalata B1 .
-
Membrane binding: Surface plasmon resonance (SPR) studies confirm affinity for phospholipid bilayers, suggesting nonspecific membrane disruption .
Comparative activity profiles:
| Cyclotide | Source | Key Activity | Mechanism |
|---|---|---|---|
| vhr1 | V. hederacea | Insecticidal, membrane binding | Membrane pore formation |
| Cter M | C. ternatea | Insecticidal, low hemolysis | Membrane disruption |
| kB1 | O. affinis | Uterotonic, anti-HIV | Receptor interaction |
Potential Applications of Recombinant vhr1
Recombinant production of vhr1 could enable:
-
Agricultural biotechnology: Engineered into crops for pest resistance, leveraging its insecticidal properties .
-
Pharmaceutical design: As a stable scaffold for peptide-based drugs, benefiting from its low hemolytic activity compared to kB1 .
Research Gaps and Future Directions
While native vhr1’s structure and activity are well-characterized , recombinant expression systems for large-scale production remain underexplored. Further studies should address:
-
Optimization of heterologous expression in microbial or plant hosts.
-
Structure-activity relationship (SAR) studies to enhance target specificity.
Product Specs
Target Background
Q&A
Experimental Design for Recombinant Cyclotide Production
Q: What experimental design considerations are crucial for producing recombinant Viola hederacea root cyclotide 1? A: When designing experiments for recombinant cyclotide production, consider the following:
-
Expression System: Choose an appropriate host organism (e.g., E. coli or yeast) based on the desired yield and ease of purification.
-
Gene Construction: Ensure the cyclotide gene is correctly inserted into an expression vector with suitable promoters and terminators.
-
Purification Methods: Develop efficient purification protocols to isolate the recombinant cyclotide, such as affinity chromatography or HPLC.
Structural Analysis of Cyclotides
Q: How can the structural integrity of recombinant Viola hederacea root cyclotide 1 be verified? A: Use techniques like NMR spectroscopy to confirm the cyclized backbone and cystine knot motif, which are characteristic of cyclotides. Mass spectrometry can also verify the molecular weight and confirm the presence of disulfide bonds .
Biological Activity Assays
Q: What methods can be employed to assess the biological activity of recombinant cyclotides? A: Biological activity can be evaluated through:
-
Insecticidal Activity: Use bioassays against insect pests to assess membrane disruption capabilities.
-
Antimicrobial Activity: Test against various pathogens to determine efficacy.
-
Antiviral Activity: Conduct cell-based assays to evaluate activity against viruses like HIV .
Data Contradiction Analysis in Cyclotide Research
Q: How can researchers address discrepancies in data regarding cyclotide activity or structure? A: Address data contradictions by:
-
Repeating Experiments: Verify results through replication.
-
Methodological Review: Ensure consistency in experimental methods.
-
Comparative Analysis: Compare findings with existing literature to identify potential sources of variation.
Tissue-Specific Expression of Cyclotides
Q: What insights can be gained from studying tissue-specific expression of cyclotides in Viola hederacea? A: Tissue-specific expression studies reveal compartmentalized defense mechanisms in plants. For example, roots may express different cyclotides than leaves, reflecting adaptation to diverse pathogens .
Therapeutic Potential of Cyclotides
Q: How can cyclotides like Viola hederacea root cyclotide 1 be explored for therapeutic applications? A: Explore therapeutic potential by:
-
Drug Design Templates: Utilize cyclotides as scaffolds for designing novel drugs due to their stability and bioactivity.
-
Pharmaceutical Applications: Investigate their use in treating diseases such as HIV or as antimicrobial agents .
Advanced Research Questions in Cyclotide Biology
Q: What are some advanced research questions in cyclotide biology that could guide future studies? A: Advanced research questions include:
-
Evolutionary Origins: Investigate how cyclotides evolved in different plant families.
-
Mechanisms of Action: Elucidate the molecular mechanisms underlying cyclotide bioactivity.
-
Synergistic Effects: Explore potential synergies between cyclotides and other plant defense peptides .
Methodological Challenges in Cyclotide Research
Q: What methodological challenges are commonly encountered in cyclotide research, and how can they be addressed? A: Common challenges include:
-
Purification Complexity: Overcome by optimizing chromatography conditions.
-
Structural Analysis: Use advanced spectroscopic techniques to confirm structure.
-
Activity Assays: Develop robust bioassays to accurately measure biological activity .
Example Data Table: Cyclotide Characteristics
| Cyclotide | Plant Source | Biological Activity | Structural Features |
|---|---|---|---|
| Vhr1 | Viola hederacea (Root) | Insecticidal, antimicrobial | Cystine knot motif, three disulfide bonds |
| Kalata B1 | Oldenlandia affinis | Insecticidal, anthelmintic | CCK motif, highly stable |
| Cter M | Clitoria ternatea | Insecticidal, membrane disruption | Embedded in albumin precursor |
