Recombinant Lupinus luteus Protein PR-L1

How can researchers differentiate between PR protein classes in yellow lupin?

Researchers should employ a combination of molecular and biochemical approaches to differentiate PR protein classes. First, conduct protein extraction followed by two-dimensional gel electrophoresis to separate proteins based on isoelectric point and molecular weight. Subsequently, perform Western blot analysis using specific antibodies against PR10 proteins, as demonstrated with anti-LlPR10.1AN antibody, which can confirm immunochemical properties and distinguish between native and recombinant forms . For definitive classification, mass spectrometry analysis should be employed, similar to the approach used in conglutin protein studies, where targeted assays with multiple reaction monitoring (MRM) transitions allowed for protein differentiation based on unique peptide signatures .

What is known about PR protein induction in response to pathogens?

While the search results don't specifically detail PR10 protein induction patterns, research on defense mechanisms in Lupinus luteus against Colletotrichum lupini provides valuable insights. Defense responses in resistant plants involve the activation of phenylpropanoid and salicylic acid pathways, generation of reactive oxygen species (ROS), and hypersensitive response at infection sites . Although this research focused on R proteins rather than PR proteins, it suggests that PR proteins might be similarly regulated during pathogen challenge, as both are components of the plant immune system. Researchers should investigate PR protein expression patterns at different time points post-infection (e.g., 12, 24, 60, and 84 hours) to establish temporal induction profiles.

Which expression system yields optimal results for Lupinus luteus PR protein production?

The Escherichia coli expression system using pET-series plasmids has proven effective for producing substantial quantities of Lupinus luteus PR10.1A protein. Specifically, both pET-3a and pET-15b vectors carrying the T7 promoter were successfully used for overexpression in E. coli strain BL21(DE3)pLysS . The choice between these two vectors depends on downstream applications:

Researchers should select the expression system based on their specific experimental requirements and downstream applications .

What strategies can optimize solubility of recombinant Lupinus luteus PR proteins?

Solubility optimization for recombinant Lupinus luteus PR proteins requires a multifaceted approach. When using the pET-3a system, the LlPR10.1A protein forms insoluble inclusion bodies that require renaturation. To improve solubility:

• Adjust expression conditions: Lower induction temperature (16-20°C), reduce IPTG concentration, and employ slower induction protocols.

• Modify buffer composition during purification: Test different pH values, salt concentrations, and additives like glycerol or mild detergents.

• Consider fusion partners: Although not specifically mentioned for PR proteins, fusion tags like MBP (maltose-binding protein) or SUMO might enhance solubility.

• Optimize renaturation protocols: For proteins expressed as inclusion bodies, a gradual dialysis approach removing denaturants can improve recovery of properly folded proteins.

The pET-15b system produces His-tagged protein that remains soluble at moderate concentrations but precipitates above 10 mg/ml, suggesting inherent solubility limitations that require careful buffer optimization .

How do post-translational modifications differ between native and recombinant PR proteins?

• Mass spectrometry analysis of both native and recombinant proteins to identify and quantify PTMs.

• Functional assays to determine if any activity differences exist between native and recombinant forms.

• Structural studies (X-ray crystallography or NMR) to detect structural variations that might result from differential PTMs.

These approaches would provide a comprehensive comparison of native versus recombinant PR protein characteristics.

What purification protocol yields highest purity for recombinant PR proteins from Lupinus luteus?

The purification strategy depends on the expression system used. For LlPR10.1A protein expressed in the pET-3a system, a multi-step protocol achieved homogeneity:

• Ammonium sulfate fractionation of insoluble inclusion bodies

• Ion-exchange chromatography on DE 52 cellulose

• Size exclusion chromatography on Superdex 75 FPLC column

For His-tagged LlPR10.1A protein expressed using the pET-15b system, a simplified approach using affinity chromatography was effective:

• Direct purification using Ni²⁺-charged His-Bind Resin

Both methods yielded proteins with identical immunochemical properties as determined by Western blot analysis. Researchers should select the purification strategy based on available equipment, desired purity levels, and downstream applications.

How can researchers validate the structural integrity of purified recombinant PR proteins?

To validate structural integrity of purified recombinant PR proteins, researchers should implement a multi-analytical approach:

• Immunological validation: Perform Western blot analysis with specific antibodies (like anti-LlPR10.1AN) to confirm immunochemical identity with native proteins .

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Thermal shift assays to determine protein stability

  • Dynamic light scattering to evaluate homogeneity and aggregation state

• Functional validation: Develop activity assays based on hypothesized functions (e.g., ribonuclease activity reported for some PR10 proteins).

• Structural analysis: For definitive validation, X-ray crystallography (the renatured protein from pET-3a system concentrated to 22 mg/ml was suitable for crystallization attempts) .

This comprehensive validation strategy ensures both structural and functional integrity of the purified recombinant protein.

What analytical techniques are most effective for quantifying PR protein expression levels?

For accurate quantification of PR protein expression levels, researchers should employ:

• Targeted mass spectrometry: Develop multiple reaction monitoring (MRM) methods similar to those described for conglutin proteins . This approach provides:

  • High specificity through monitoring of unique peptide signatures

  • Accurate quantification using multiple transitions per peptide

  • Capability to distinguish between highly similar protein isoforms

• Immunoassays:

  • ELISA using specific antibodies for high-throughput quantification

  • Western blot with densitometry for semi-quantitative analysis

• Spectroscopic methods:

  • UV absorbance at 280 nm (corrected for the specific extinction coefficient)

  • Colorimetric protein assays (Bradford, BCA) calibrated with purified PR protein standards

For optimal accuracy, researchers should calibrate their preferred method using purified recombinant PR protein standards of known concentration and validate results using complementary techniques.

What structural features distinguish Lupinus luteus PR10 proteins from other PR protein families?

Although the specific structural features of Lupinus luteus PR10 proteins are not explicitly detailed in the search results, researchers can draw insights from general PR10 protein characteristics and the crystallization efforts described. PR10 proteins typically feature:

• A conserved P-loop motif (phosphate-binding loop)

• A glycine-rich region

• A hydrophobic cavity that can bind various ligands, potentially including plant hormones or secondary metabolites

The successful concentration of renatured LlPR10.1A protein to 22 mg/ml for crystallization suggests a stable, well-folded structure amenable to structural studies. Researchers should employ X-ray crystallography or NMR spectroscopy to elucidate the specific structural features of Lupinus luteus PR10 proteins and compare them with PR10 proteins from other plant species to identify conserved and unique structural elements.

How can researchers investigate potential enzymatic activities of PR proteins?

To investigate potential enzymatic activities of PR proteins from Lupinus luteus, researchers should implement a systematic approach:

• Computational prediction: Perform sequence and structural homology analysis to identify potential enzymatic domains or active sites.

• Substrate screening:

  • Test for ribonuclease activity, a common function in some PR10 proteins

  • Assess potential phospholipase activity

  • Investigate binding to potential ligands (hormones, secondary metabolites)

• Activity assays:

  • Develop spectrophotometric or fluorometric assays for real-time monitoring

  • Implement radiolabeled substrate assays for high sensitivity

  • Utilize mass spectrometry to identify reaction products

• Structure-function studies:

  • Conduct site-directed mutagenesis of predicted active site residues

  • Perform crystallography with substrate analogs to identify binding sites

This comprehensive approach would help establish the enzymatic functions of Lupinus luteus PR proteins and their potential roles in plant defense mechanisms.

What experimental approaches can reveal PR protein interactions with pathogen molecules?

To investigate PR protein interactions with pathogen molecules, researchers should employ a combination of in vitro and in vivo approaches:

• Protein-protein interaction studies:

  • Pull-down assays using recombinant PR proteins as bait

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Pathogen challenge experiments:

  • Co-localization studies during infection using fluorescently labeled PR proteins

  • In planta protein crosslinking followed by mass spectrometry

  • Yeast two-hybrid screening against pathogen protein libraries

• Functional validation:

  • Competitive binding assays with purified pathogen components

  • In vitro inhibition assays against pathogen growth or virulence factors

  • Transgenic plants with modified PR protein expression to assess resistance phenotypes

These approaches would provide comprehensive insights into the molecular mechanisms by which PR proteins contribute to plant defense against pathogens.

How can recombinant PR proteins advance our understanding of plant immunity mechanisms?

Recombinant PR proteins from Lupinus luteus serve as valuable tools for investigating plant immunity through several applications:

• Structure-function studies: Purified recombinant proteins enable determination of three-dimensional structures, providing insights into functional domains and potential binding sites.

• Protein-protein interaction networks: Use as bait in interactome studies to identify plant proteins that interact with PR proteins during immune responses.

• Development of molecular markers: Generation of specific antibodies against recombinant PR proteins facilitates monitoring of PR protein accumulation during pathogen infection .

• Comparative functional genomics: Comparing Lupinus luteus PR proteins with homologs from other plant species can reveal conserved and species-specific defense mechanisms.

• Mechanistic studies: Investigating how PR proteins interface with other components of plant defense, such as R proteins like LluR1 that trigger effector-triggered immunity (ETI) and hypersensitive response .

By leveraging recombinant PR proteins as molecular tools, researchers can unravel the complex signaling networks underpinning plant immunity.

How do PR proteins integrate with the broader plant immune network in Lupinus luteus?

Understanding how PR proteins integrate with the broader immune network requires contextualizing them within established defense mechanisms in Lupinus luteus:

• Relationship with R protein-mediated resistance: While PR proteins and R proteins like LluR1 both contribute to immunity, they operate at different levels. LluR1 encodes a TIR-NBS-LRR protein that functions as an immune receptor triggering effector-triggered immunity (ETI) , while PR10 proteins may function downstream or in parallel pathways.

• Signaling pathway integration: Research on Lupinus luteus defense against Colletotrichum lupini indicates that resistance involves activation of salicylic acid and phenylpropanoid pathways . PR proteins might be regulated by these pathways or contribute to their activation.

• ROS and hypersensitive response: Resistant Lupinus luteus plants exhibit ROS generation at infection sites and hypersensitive response that constrains pathogen proliferation . Researchers should investigate whether PR proteins influence or respond to ROS signaling.

• Temporal dynamics: Defense responses show distinct temporal patterns, with differential gene expression at various time points post-infection (12, 24, 60, and 84 hours) . Understanding when PR proteins are expressed relative to these time points would clarify their position in the defense response sequence.

This integrated view would position PR proteins within the broader context of Lupinus luteus immunity.

What challenges remain in translating PR protein research to enhanced crop protection strategies?

Several challenges must be addressed to translate PR protein research into effective crop protection strategies:

• Functional characterization gap: Despite identification of PR proteins like LlPR10.1A and LlPR10.1B, their precise contribution to plant defense remains unknown . Establishing clear molecular functions is essential for leveraging these proteins in crop protection.

• Pathogen specificity understanding: Research must determine whether PR proteins provide broad-spectrum or pathogen-specific protection. Current research on Lupinus luteus focuses on specific pathogens like Colletotrichum lupini , but understanding PR protein efficacy against diverse pathogens is crucial.

• Integration with breeding programs: Connecting PR protein expression patterns with genetic markers would facilitate breeding for enhanced disease resistance. The identification of resistance genes like LluR1 provides a model for how PR protein research could be similarly integrated.

• Environmental stability factors: For field application, researchers must determine how environmental factors affect PR protein stability and function, similar to how protein isolate properties have been characterized under different conditions .

• Transgenic approaches: Developing transgenic strategies for optimal PR protein expression patterns requires careful consideration of expression timing, tissue specificity, and potential metabolic costs to the plant.

Addressing these challenges would facilitate translation of fundamental PR protein research into practical crop protection strategies.