Recombinant Erythrina variegata Trypsin inhibitor 1B
Description
Overview of Erythrina Trypsin Inhibitors
Trypsin inhibitors from Erythrina species belong to the Kunitz-type family, characterized by their compact structure stabilized by disulfide bonds. These inhibitors typically exhibit:
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Molecular weights: ~19–21 kDa (e.g., E. velutina EvTI: 19,210.48 Da ).
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Mechanism: Non-competitive inhibition with low IC50 values (e.g., EvTI: IC50 = 2.2 × 10⁻⁸ mol·L⁻¹ for trypsin ).
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Functional stability: Dependent on disulfide bonds, which confer resistance to pH extremes and chaotropic agents .
2.1. Primary Structure
Partial sequencing of E. velutina EvTI revealed homology to other legume Kunitz inhibitors, with conserved residues critical for protease binding (Table 1) .
Table 1: Sequence alignment of Kunitz-type inhibitors
| Inhibitor | N-terminal Sequence | Source |
|---|---|---|
| EvTI (E. velutina) | GSGHRHESTDEPSSSKAACCD | Machado et al., 2013 |
| ETI (E. caffra) | GSGHRHESTDEPSSSKAACCD | Dzoyem et al., 2014 |
| BvvTI (Bauhinia spp.) | –NHHDSDSSDEPSESSEPCCDSCICSKSIPPQCHHTDIRL | Fang et al., 2010 |
2.2. Key Functional Domains
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Reactive site: Arg and Ser residues mediate trypsin binding .
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Disulfide bonds: Essential for stability but not inhibitory activity .
3.1. Expression Systems
While no studies on recombinant E. variegata inhibitors were found, E. caffra ETI has been successfully refolded after recombinant expression, retaining native structure and activity . Key steps include:
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Reduction/oxidation: Glutathione buffers restore disulfide bonds .
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Chromatography: Affinity (trypsin-Sepharose) and RP-HPLC for purification .
3.2. Pharmacological Activities
Recombinant Kunitz inhibitors from related species demonstrate:
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Anticoagulant effects: Prolonged activated partial thromboplastin time (APTT) in murine models .
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Anti-inflammatory action: Reduced TNF-α and IL-6 levels, increased IL-12 and IFN-α in sepsis models .
Table 2: Pharmacological parameters of E. velutina EvTI
| Activity | Value/Outcome | Model |
|---|---|---|
| Trypsin inhibition (Ki) | 1.0 × 10⁻⁸ mol·L⁻¹ | In vitro assay |
| APTT prolongation | 2-fold increase at 1 mg·kg⁻¹ | Murine sepsis |
| Leukocyte migration | 60% reduction | Peritoneal fluid |
Toxicity and Clinical Potential
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Safety: No recorded drug interactions or toxicity in preclinical studies .
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Therapeutic targets: Apoptosis induction in cancer cells via caspase activation , insulin signaling modulation via PTP1B inhibition .
Research Gaps and Future Directions
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Structural studies: X-ray crystallography of rEvTI-1B to resolve binding motifs.
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Clinical trials: Efficacy and safety profiling in metabolic or oncological models.
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Optimization: Codon-optimized expression in E. coli or yeast for scalable production.
Product Specs
Target Background
Q&A
What are the structural characteristics of Recombinant Erythrina variegata Trypsin Inhibitor 1B (ETIb)?
Recombinant Erythrina variegata Trypsin Inhibitor 1B (ETIb) is a Kunitz-type trypsin inhibitor. It consists of 176 amino acid residues with a molecular weight of approximately 19,783 Da. ETIb shares significant structural homology with other Kunitz-type inhibitors, such as soybean trypsin inhibitor (STI), and exhibits about 65% amino acid sequence identity with its counterpart, ETIa .
What experimental methods are used to purify and characterize recombinant ETIb?
Recombinant ETIb can be expressed in E. coli and purified using gel filtration chromatography on Sephadex G-75, followed by reverse-phase HPLC. Characterization involves SDS-PAGE for purity assessment and Western blotting for confirmation of the recombinant protein .
How does the amino acid sequence of ETIb compare to other Kunitz-type inhibitors?
ETIb shares about 45% identical residues with soybean trypsin inhibitor (STI), characteristic of Kunitz-type inhibitors. It also exhibits homology with storage proteins like sporamin from sweet potato and miraculin from miracle fruit, with about 30% identical residues .
How can site-directed mutagenesis be used to study ETIb's inhibitory mechanisms?
Site-directed mutagenesis can be employed to alter specific amino acids in ETIb, such as those at the reactive site, to study their role in inhibiting target enzymes. This approach helps elucidate the structural basis of ETIb's inhibitory activity and can inform design of modified inhibitors with enhanced specificity or potency .
What are the challenges in expressing recombinant ETIb in heterologous systems?
Expressing recombinant ETIb in systems like E. coli may result in the addition of extra amino acids at the N-terminus, which can affect the protein's inhibitory activity. Ensuring proper folding and removal of these extra residues is crucial for maintaining the protein's native function .
How does the inhibitory activity of ETIb compare to that of ETIa?
ETIb and ETIa both inhibit plasmin but differ in their ability to inhibit other serine proteinases. ETIa inhibits tissue-type plasminogen activator (tPA) and plasma kallikrein, whereas ETIb does not. This difference highlights the specificity of these inhibitors in biological systems .
What analytical techniques are used to assess the purity and yield of recombinant ETIb?
Techniques such as SDS-PAGE and reverse-phase HPLC are used to assess the purity of recombinant ETIb. The yield can be quantified by measuring the protein concentration after purification steps .
How can the findings from ETIb research contribute to understanding proteinase inhibitor functions in plants?
Studies on ETIb contribute to understanding the role of Kunitz-type inhibitors in plant defense mechanisms and their potential applications in medicine. These inhibitors can serve as models for designing novel therapeutic agents targeting specific serine proteinases .
Data Table: Characteristics of ETIa and ETIb
| Characteristics | ETIa | ETIb |
|---|---|---|
| Amino Acid Residues | 172 | 176 |
| Molecular Weight (Da) | 19,242 | 19,783 |
| Sequence Identity with ETIb | 65% | - |
| Inhibition of Plasma Kallikrein | Yes | No |
| Inhibition of tPA | Yes | No |
| Inhibition of Plasmin | Yes | Yes |
