Recombinant Vegetative protein 16

What is Recombinant Vegetative protein 16 and what are its key structural characteristics?

Recombinant Vegetative protein 16 refers to the artificially expressed form of PI-16 (Peptidase Inhibitor 16), a putative serine protease inhibitor. The protein has a molecular weight of approximately 46 kDa including tags when expressed in mammalian systems . The human variant contains multiple functional domains and is expressed naturally in prostate, testis, ovary, and intestinal tissues .

Key structural features include:

• A protein fragment spanning residues 28 to 442

• C-terminal DDDDK tag in recombinant versions

• Multiple domains including protease inhibitory regions

What expression systems are most effective for Recombinant Vegetative protein 16 production?

Several expression systems have been validated for recombinant protein production, each with specific advantages depending on research objectives:

HEK 293 cells have been specifically documented for successful expression of human PI-16 , while yeast expression systems like Pichia pastoris have been effective for other recombinant proteins as demonstrated with Ixodes scapularis Calreticulin .

How can expression vectors be optimized for maximum Recombinant Vegetative protein 16 yield?

Research indicates that combinatorial approaches significantly enhance recombinant protein expression. For instance:

• Promoter stacking strategy: Multiple constitutive and tissue-specific promoters can increase expression by 20-fold compared to single promoter systems .

• Vector design considerations:

  • Use of appropriate signal sequences for secretion

  • Codon optimization for the host expression system

  • Inclusion of appropriate fusion tags for enhanced solubility and purification

• Event stacking approach: Re-transformation of already transformed lines with additional expression vectors can further boost protein accumulation (demonstrated to increase from 1.4% to 11.5% of total soluble protein) .

What methods are recommended for purification of Recombinant Vegetative protein 16?

Based on reported protocols for similar recombinant proteins:

• Affinity chromatography: For tagged PI-16 variants, such as those with a C-terminal DDDDK tag, appropriate affinity columns provide high specificity .

• Sequential purification protocol:

  • Initial concentration by ammonium sulfate precipitation (as demonstrated with rIxsCRT)

  • Dialysis against appropriate column-binding buffer (e.g., 1 M NaCl, 0.4 M Tris, 0.2 M imidazole, pH 7.4)

  • Affinity purification using HiTrap chelating HP columns or equivalent

  • Final dialysis against application-appropriate buffer (10 mM HEPES, pH 7.4; 1× PBS, pH 7.4; or normal saline)

• Purity assessment: SDS-PAGE analysis with silver staining and Western blotting using tag-specific antibodies can confirm purification success .

What techniques are effective for studying interaction partners of Recombinant Vegetative protein 16?

Several complementary approaches have been validated for studying protein-protein interactions:

• DiffPOP (Differential Precipitation of Proteins): This technique has successfully identified multiple interaction partners for recombinant proteins . The procedure involves:

  • Co-incubation of recombinant protein with potential binding partners

  • Serial precipitation using increasing concentrations of ammonium sulfate

  • Analysis of co-precipitating fractions by Western blotting

  • Identification of interacting proteins by LC-MS/MS

• Pull-down assays: Can validate specific interactions between the recombinant protein and candidate binding partners .

• Mass spectrometry-based approaches: LC-MS/MS analysis has identified over 1000 unique interactions for similar recombinant proteins (e.g., rIxsCRT interactions with 1074-1936 unique proteins across different precipitation fractions) .

How can the biological activity of Recombinant Vegetative protein 16 be assessed?

Functional characterization requires multiple approaches:

• Enzyme inhibition assays: Since PI-16 is a putative serine protease inhibitor, assays measuring inhibition of target proteases (using chromogenic or fluorogenic substrates) can quantify activity .

• Co-culture experiments: Similar to approaches used with other recombinant proteins, co-culture with relevant cellular systems can reveal biological effects. For example, monitoring growth patterns or morphological changes in the presence of the recombinant protein .

• Reactome analysis: For proteins with multiple binding partners, reactome analysis can identify enriched pathways and potential biological functions. This approach has been used successfully with other recombinant proteins to characterize over 1.5-fold higher interactions compared to controls .

How can immunogenic properties of Recombinant Vegetative protein 16 be characterized?

Immunoproteomics approaches provide comprehensive characterization:

• 2D gel electrophoresis followed by western blotting: This technique has been effective for identifying immunogenic proteins in complex mixtures, as demonstrated with Bacillus anthracis spore and vegetative proteins .

• Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): This technique can identify immunogenic spots from 2D gels after trypsin digestion .

• Verification of immunogenicity: Expression of identified proteins and subsequent western blotting with immune sera can confirm immunogenicity of specific proteins .

What approaches can be used to enhance stability of purified Recombinant Vegetative protein 16?

Optimal storage and handling conditions include:

• Short-term storage: Store at +4°C for up to 1-2 weeks .

• Long-term storage:

  • Aliquot to minimize freeze-thaw cycles

  • Store at -80°C

  • Avoid repeated freeze-thaw cycles which can decrease activity

• Reconstitution protocol: For lyophilized protein, add deionized water to prepare a working stock solution of 0.5 mg/mL and ensure complete dissolution. Filter through an appropriate sterile filter before cell culture applications .

How can issues with expression levels be addressed?

When facing low expression yields:

• Vector system optimization: Consider using viral-based vectors like the magnICON system for plant-based expression or deconstructed vectors containing viral elements that allow transgene spread throughout the plant .

• Host system selection:

  • For non-tobacco plants, geminiviral vectors with broad host ranges may be more suitable than tobacco mosaic virus (TMV)-based vectors

  • For proteins with heterosubunits, avoid vectors with competing replicons (such as magnICON), as cells will typically only amplify one vector

• Expression conditions: Optimize temperature, induction time, and media composition based on the specific properties of PI-16.

What strategies can improve solubility of Recombinant Vegetative protein 16?

Solubility enhancement approaches include:

• Fusion partner selection: Choose solubility-enhancing fusion tags appropriate for the expression system (DDDDK tag has been used successfully for human PI-16) .

• Buffer optimization: Test various buffer compositions during extraction and purification to maximize stability and solubility.

• Co-expression strategies: For complex proteins, co-expression with chaperones or binding partners may improve folding and solubility.