Recombinant Solanum tuberosum Guanine nucleotide-binding protein subunit beta (GB1)

What is the structure and function of GB1 when used in potato recombinant protein systems?

GB1 is a small domain (~56 amino acids) derived from Streptococcal protein G that can be fused to target proteins to enhance their expression and stability. When fused to the N-terminus of various target proteins in potato, GB1 significantly improves expression levels by 1.3- to 3.1-fold at the protein level depending on the specific target . The domain functions through enhancing both transcriptional and translational efficiency of fusion proteins. Studies using qRT-PCR analysis have demonstrated that GB1-GFP shows a 1.7-fold increase in transcript levels compared to GFP alone when expressed in Nicotiana benthamiana . Additionally, analysis using wheat germ extracts in vitro translation systems showed that GB1-LUC constructs increased luminescence signals by 1.6-fold at 60 minutes and 2.0-fold at 120 minutes compared to LUC alone, confirming GB1's positive effect on translation rates .

How does GB1 fusion affect protein localization in plant systems?

The effect of GB1 on increasing protein expression is independent of subcellular localization. Research has demonstrated that GB1 fusion enhances protein expression in multiple cellular compartments including the endoplasmic reticulum (ER), chloroplasts, and cytosol. Experiments with GFP fusion proteins showed that BiP-GB1-GFP (ER-targeted), RbcS(tp)-GB1-GFP (chloroplast-targeted), and cytosolic GB1-GFP all exhibited significantly higher fluorescence signals compared to their respective non-GB1 counterparts at 3 days post-infiltration . The signal intensity ratios between GB1-GFP and GFP were approximately 2.5, 2.5, and 1.7 for ER-, chloroplast-, and cytosol-localized proteins respectively, indicating that GB1's beneficial effects operate across different cellular compartments .

How does GB1 differ from other fusion tags when used in potato expression systems?

Unlike many larger fusion tags that can interfere with protein function or require complex removal procedures, GB1 offers several unique advantages:

GB1 uniquely enables direct detection of fusion proteins using secondary anti-IgG antibodies without requiring primary antibodies for western blot analysis, which simplifies detection protocols and reduces experimental costs .

What are the optimal expression systems for producing GB1-fusion proteins in potato?

For transient expression in potato systems, Agrobacterium-mediated infiltration has proven most effective for GB1-fusion proteins. The optimal protocol involves:

• Constructing vectors with GB1 fused to the N-terminus of the target protein

• Including appropriate subcellular targeting sequences if desired (e.g., BiP leader sequence for ER targeting)

• Using Agrobacterium strain GV3101 transformed with the expression construct

• Infiltrating Nicotiana benthamiana leaves at OD600 of 0.4-0.6

• Harvesting tissue 3-7 days post-infiltration, with peak expression typically observed at 3 days

For stable transformation, similar vector designs can be used with established potato transformation protocols utilizing internodal sections co-cultivated with Agrobacterium tumefaciens, followed by a two-stage callus induction/shoot outgrowth system under appropriate selection .

What purification strategies maximize recovery of functional GB1-fusion proteins?

When purifying GB1-fusion proteins from potato or other plant tissues, the following optimized protocol is recommended:

• Harvest plant tissue (typically 0.1-0.5g) and flash-freeze in liquid nitrogen

• Grind tissue to a fine powder and extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 0.5% Triton X-100

  • 1 mM PMSF and protease inhibitor cocktail

• Clarify lysate by centrifugation (15,000 × g, 15 min, 4°C)

• For His-tagged GB1-fusion proteins, purify using Ni²⁺-NTA affinity chromatography

• For proteins without additional tags, leverage GB1's IgG-binding properties using IgG-Sepharose affinity chromatography

• Consider a two-step purification approach similar to that used for potato apyrase, involving initial affinity chromatography followed by Cibacron Blue-affinity chromatography for highest purity

This approach has shown effective recovery of functional GB1-fusion proteins with yields typically ranging from 0.5-5 mg per liter of plant culture equivalent .

What methods are effective for removing the GB1 tag while preserving protein function?

For applications requiring GB1 tag removal, incorporating a protease cleavage site between GB1 and the target protein is essential. Research has demonstrated successful removal using the tobacco etch virus (TEV) protease site:

• Design constructs with a TEV protease recognition sequence (ENLYFQ↓G) between GB1 and the target protein

• Purify the GB1-fusion protein using appropriate affinity methods

• Treat with TEV protease at either 10°C or 25°C overnight

• Separate cleaved target protein from GB1 and uncleaved fusion proteins using reverse affinity chromatography

This approach has been successfully demonstrated with GB1-CTB fusion proteins, where most of the fusion protein was effectively cleaved by TEV protease treatment at both 10°C and 25°C conditions . The released target protein showed the expected reduction in molecular weight while maintaining functional integrity.

How can GB1 be used as a tool to study G-protein signaling in potato?

GB1 fusion constructs provide an excellent platform for studying G-protein signaling pathways in potato through:

• Protein-protein interaction studies: By creating GB1 fusions with G-protein subunits (such as StGNB3) or their interacting partners, researchers can perform pull-down assays to identify novel interaction partners in potato signaling networks .

• Subcellular localization analysis: GB1-GFP fusions with G-protein components allow visualization of protein localization and trafficking in living plant cells.

• Functional complementation: In potatoes with mutations in G-protein components (like the mutation in GNB3 that causes retinal degeneration in chickens ), GB1-fused wild-type proteins can be used to assess functional rescue.

• Structure-function studies: By introducing specific mutations in GB1-fused G-protein components, researchers can assess the impact on function while maintaining high expression levels. This approach has been used successfully to study mutations in the KTGGL-encoding locus using cytidine base editors .

What experimental approaches can be used to investigate GB1's role in enhancing protein expression?

To investigate the mechanistic basis of GB1's expression-enhancing effects, researchers can employ the following methodologies:

• Transcriptional analysis: Quantify transcript levels of GB1-fusion genes versus non-fused genes using qRT-PCR, as demonstrated in studies showing 1.7-fold higher transcript levels for GB1-GFP compared to GFP alone .

• Translational efficiency assessment: Use in vitro translation systems (such as wheat germ extracts) with luciferase reporters to measure translation rates, revealing a 1.6- to 2.0-fold increase in translation efficiency for GB1-fused constructs .

• Protein stability analysis: Conduct pulse-chase experiments or cycloheximide treatment followed by western blotting to determine protein half-life differences between GB1-fused and non-fused proteins.

• Structural analysis: Employ circular dichroism spectroscopy or thermal shift assays to assess folding efficiency and stability of purified GB1-fusion proteins versus non-fused controls.

• Deletion and mutation analysis: Create a series of GB1 variants with specific mutations or deletions to identify key residues responsible for the expression-enhancing effect.

How can GB1 be utilized in genome editing applications for potato improvement?

GB1 can significantly enhance CRISPR-Cas9 and base editing applications in potato through:

• Improved expression of editing components: Fusing GB1 to Cas9 or base editors can enhance their expression in potato, potentially increasing editing efficiency. This is particularly valuable in tetraploid potato where achieving edits across all four alleles is challenging .

• Enhanced delivery systems: GB1-fusion with cell-penetrating peptides or other delivery components may improve the introduction of editing machinery into plant cells.

• Target validation: Using GB1-fused proteins to complement knockout phenotypes can confirm gene function after editing, as demonstrated in studies of the StGBSSI gene in potato .

• Base editing applications: GB1 can be used to enhance expression of cytidine base editors (CBEs) that have been successfully employed to introduce precise nucleotide substitutions in potato, such as those targeting catalytic motifs of the StGBSSI enzyme .

The successful application of these techniques has been demonstrated in the precise editing of the StGBSSI gene in tetraploid potato using both traditional CRISPR-Cas9 and base editing strategies, leading to plants with impaired amylose biosynthesis .

What controls should be included when studying GB1-fusion proteins in potato systems?

When designing experiments using GB1-fusion proteins in potato, the following controls are essential:

• Non-fused target protein: Always include the target protein without GB1 fusion to directly compare expression levels, stability, and function.

• Alternative subcellular localizations: When studying localization-dependent effects, include constructs targeting the same protein to different compartments (e.g., ER, chloroplast, cytosol) with and without GB1 fusion .

• Position controls: Include both N-terminal and C-terminal GB1 fusions, as research shows the effect is strictly dependent on N-terminal localization of GB1 .

• Empty vector controls: Include empty vector transformations when assessing phenotypic effects of GB1-fusion proteins.

• Alternative tag controls: When possible, compare GB1 to other common fusion tags (His, GST, MBP) to benchmark relative performance.

• Time-course sampling: As GB1's effects on protein expression may vary over time, sample across multiple time points (3, 5, and 7 days post-infiltration is recommended based on previous studies) .

How should researchers design GB1 fusion constructs for optimal expression in potato?

For optimal results when designing GB1 fusion constructs:

• Fusion orientation: Position GB1 at the N-terminus of the target protein, as C-terminal fusions do not demonstrate the same enhancement effect .

• Linker selection: Include a flexible linker (such as GGGGS) between GB1 and the target protein to minimize steric hindrance.

• Codon optimization: Optimize the GB1 coding sequence for expression in potato, as demonstrated in successful studies with other recombinant proteins .

• Vector selection: Use plant expression vectors with strong constitutive promoters (e.g., CaMV 35S) or tissue-specific promoters as appropriate for the research question.

• Targeting signals: If subcellular targeting is desired, position targeting sequences (such as the BiP leader sequence for ER targeting) upstream of the GB1 domain .

• Cleavage sites: Include a protease recognition sequence (e.g., TEV site) between GB1 and the target protein if tag removal will be necessary .

• Additional tags: Consider incorporating C-terminal purification tags (His6) to facilitate two-step purification when highest purity is required .

Why might GB1-fusion proteins show unexpectedly low expression in potato systems?

When GB1-fusion proteins exhibit lower than expected expression levels, consider these potential issues and solutions:

Systematic evaluation of each potential issue can help identify and resolve the specific cause of reduced expression.

How can researchers address protein aggregation issues with GB1-fusion proteins?

When working with GB1-fusion proteins that show aggregation tendencies:

• Optimize extraction conditions:

  • Test buffer pH ranges (6.5-8.0)

  • Evaluate different salt concentrations (100-500 mM NaCl)

  • Include mild detergents (0.1% Triton X-100 or NP-40)

  • Add stabilizing agents (5-10% glycerol, 1-5 mM DTT, 0.5M arginine)

• Modify expression parameters:

  • Reduce expression temperature by growing plants at 18-22°C

  • Harvest earlier (2-3 days post-infiltration instead of 5-7)

  • Use weaker promoters to reduce expression rate

• Adjust protein design:

  • Incorporate solubility-enhancing tags in addition to GB1

  • Modify the linker length or composition

  • Remove hydrophobic regions from the target protein if possible

• Processing adjustments:

  • Perform all purification steps at 4°C

  • Consider on-column refolding during purification

  • Filter solutions through 0.22 μm filters before chromatography

These approaches have proven effective in resolving aggregation issues with challenging proteins in plant expression systems .

How can CRISPR-Cas9 technology be applied to study GB1 function in potato?

CRISPR-Cas9 technologies offer powerful approaches for investigating GB1 and G-protein signaling in potato:

• Gene knockout studies: Design sgRNAs targeting endogenous G-protein beta subunit genes to create knockout lines for functional analysis. This approach has been successfully applied to create tetra-allelic mutants in tetraploid potato .

• Base editing applications: Use cytidine base editors (CBEs) to introduce specific amino acid changes in G-protein components without creating double-strand breaks. This method has successfully induced DNA substitutions in the KTGGL-encoding locus of potato proteins .

• Screening optimization: Implement high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis for rapid, sensitive and cost-effective screening of edited plants .

• Promoter studies: Use CRISPR to edit or delete regulatory elements controlling GB1 expression to study their function.

• Tagging endogenous genes: Apply CRISPR-mediated homology-directed repair to add fluorescent or affinity tags to endogenous G-protein genes for studying their native behavior.

• Multiplexed editing: Target multiple components of G-protein signaling pathways simultaneously to study pathway interactions and redundancy.

The successful application of CRISPR in tetraploid potato represents a significant technical achievement that opens new avenues for genome engineering in this species .

What approaches are most effective for studying the role of G-proteins in potato disease resistance?

To investigate G-protein involvement in potato disease resistance:

• Gene silencing approaches: Virus-induced gene silencing (VIGS) of G-protein components can quickly assess their role in disease response without stable transformation. This approach revealed that silencing of StRab5b, a small G protein in potato, facilitated pathogen infection, confirming its role in disease resistance .

• Transient overexpression: Agrobacterium-mediated transient expression of GB1-fused G-protein components can enhance expression and reveal gain-of-function phenotypes. For example, transient expression of StRab5b in N. benthamiana enhanced resistance to P. infestans .

• Stable transgenic approaches: Generate potato lines stably expressing GB1-fused G-protein components to study long-term and developmental effects on disease resistance.

• Biochemical assays: Measure ROS production, enzyme activities, and defense hormone levels in G-protein modified plants. Research shows that stable expression of StRab5b enhanced redox-stress response capacity through H₂O₂ accumulation in infected leaves and increased activity of ROS scavenging enzymes .

• Transcriptome analysis: Compare defense-related gene expression profiles between wild-type and G-protein modified plants to identify downstream components of defense signaling.

• Protein-protein interaction studies: Use co-immunoprecipitation with GB1-fused G-proteins to identify interacting partners during pathogen infection.

These approaches have successfully demonstrated that StRab5b positively regulates resistance against potato late blight via JA-mediated defense signaling pathway .

How can researchers investigate structural dynamics of GB1 interactions using computational approaches?

Computational methods provide valuable insights into GB1 structural dynamics:

• Homology modeling: Generate structural models of potato G-protein components based on known structures from other organisms. When modeling novel GB1 fusion proteins, consider how the D153del mutation in GNB3 (which deletes a highly conserved aspartic acid residue in the third of seven WD domains) destabilizes the protein structure .

• Molecular dynamics simulations: Perform atomistic simulations of GB1 fusion proteins to assess stability, conformational changes, and potential folding pathways.

• Protein-protein docking: Model interactions between GB1-fused proteins and their binding partners to identify key interface residues.

• In silico mutagenesis: Computationally predict the effects of mutations in GB1 or target proteins on their interaction and stability.

• Binding energy calculations: Estimate the binding affinity between GB1 and target proteins or between GB1-fusion proteins and their interaction partners.

• Electrostatic surface mapping: Analyze the distribution of charges on protein surfaces to predict interaction interfaces.

These computational approaches can guide experimental design by identifying promising mutants, optimal fusion strategies, or potential interaction partners for further validation.

What statistical approaches are most appropriate for analyzing GB1 expression data?

When analyzing data from GB1 expression experiments:

• Normalization approaches:

  • For western blot data: Normalize band intensities to internal controls (housekeeping proteins)

  • For fluorescence data: Use background subtraction and normalize to total protein or cell number

  • For qRT-PCR: Apply the ΔΔCt method with appropriate reference genes stable in potato systems

• Statistical tests:

  • For comparing two conditions: Student's t-test for normally distributed data or Mann-Whitney U test for non-parametric data

  • For multiple conditions: ANOVA followed by post-hoc tests (Tukey's or Dunnett's)

  • For time-course data: Repeated measures ANOVA or mixed-effects models

• Sample size considerations:

  • Use at least 3-5 biological replicates for each condition

  • Perform power analysis to determine appropriate sample sizes for detecting expected effect sizes (1.3- to 3.1-fold changes have been observed with GB1 fusions)

• Visualization methods:

  • Bar graphs with error bars showing standard deviation or standard error

  • Box plots to show distribution of data points

  • Line graphs for time-course experiments

• Advanced analytics:

  • Principal component analysis for multi-parameter experiments

  • Regression analysis to identify correlations between protein characteristics and GB1-mediated enhancement

How should researchers interpret RNA-protein interaction data involving GB1 in potato?

When analyzing RNA-protein interactions involving GB1:

• RNA binding assay interpretation:

  • Consider both specificity and affinity in gel mobility shift assays

  • Verify binding using multiple methods (e.g., EMSA, RIP, RNA pull-down)

  • Include appropriate negative controls (non-binding RNA sequences)

  • Quantify binding curves to determine dissociation constants

• Binding motif analysis:

  • Search for enriched sequence motifs in bound RNAs

  • Pay special attention to pyrimidine-rich sequences, which are known binding sites for polypyrimidine tract-binding proteins in potato

  • Use motif discovery tools to identify novel binding sequences

• Functional validation:

  • Test mutated RNA sequences to confirm motif importance

  • Perform competition assays with synthetic RNA oligos

  • Correlate binding strength with functional outcomes in vivo

• In vivo relevance:

  • Confirm RNA-protein interactions in plant cells using techniques like CLIP-seq

  • Assess co-localization of the RNA and protein in cellular compartments

  • Determine if the interaction affects RNA stability, localization, or translation

Research has shown that polypyrimidine tract-binding proteins in potato bind to mobile RNAs like StBEL5 and enhance their stability and trafficking to select organs, demonstrating the importance of these interactions in plant development .

What bioinformatic tools are most useful for studying GB1 and G-protein signaling networks in potato?

For comprehensive analysis of GB1 and G-protein networks:

• Sequence analysis tools:

  • BLAST, Clustal Omega, and MUSCLE for sequence comparisons and alignments

  • HMMER for identifying conserved domains in G-protein sequences

  • PSIPRED and PONDR for secondary structure and disorder prediction

• Structural analysis software:

  • PyMOL, UCSF Chimera, or VMD for visualizing protein structures

  • I-TASSER, Phyre2, or AlphaFold for protein structure prediction

  • HADDOCK or ClusPro for protein-protein docking

• Network analysis platforms:

  • Cytoscape for visualizing and analyzing protein interaction networks

  • STRING database to identify known and predicted protein-protein interactions

  • KEGG and PlantReactome for pathway mapping

• Transcriptomic analysis:

  • DESeq2 or edgeR for differential expression analysis

  • WGCNA for co-expression network analysis

  • GO enrichment tools for functional annotation

• Genomic resources:

  • SpudDB and Potato Genome Sequencing Consortium resources

  • TAIR and Phytozome for comparative analysis with other plant species

  • Ensembl Plants for genome visualization and annotation

• Specialized tools:

  • NucPred or LOCALIZER for subcellular localization prediction

  • TOMTOM for motif comparison

  • ConSurf for evolutionary conservation analysis

These tools collectively enable researchers to connect molecular-level observations to system-wide understanding of G-protein signaling networks in potato.