Recombinant Burkholderia cenocepacia Bifunctional protein glk (glk)

What is the structural composition of Recombinant Burkholderia cenocepacia Bifunctional protein glk (glk)?

Recombinant Burkholderia cenocepacia Bifunctional protein glk (glk) is a full-length protein consisting of 642 amino acids (positions 1-642). When produced recombinantly, it is typically fused to an N-terminal His tag. The protein is expressed in E. coli expression systems and purified to greater than 90% purity as determined by SDS-PAGE analysis . The complete amino acid sequence is:

MSTGAQSKAVVAGQHADGPRLLADVGGTNARFALETGPGEITQIRVYPGADYPTITDAIRKYLKDVKISRVNHAAIAIANPVDGDQVTMTNHDWSFSIEATRRALGFDTLLVVNDFTALAMAL PGLTDAQRVQVGGGARRQNSVIGLLGPGTGLGVSGLIPADDRWIALGSEGGHASFAPQDEREDLVLQYARKKFPHVSFERVCAGPGMEIIYRALAARDKKRVAATVDTVEIVERAHA GDALALETVECFCGILGAFAGSVALTLGALGGVYIGGGVALKLGELFTRSSFRARFEAKGRFTHYLENIPTYLITAEYPAFLGVSAILAEQLSNRSGGASSAVFERIRQMRDALTPAERR VADLALNHPRSIINDPIVDIARKADVSQPTVIRFCRSLGCQGLSDFKLKLATGLTGTIPMSHSQVHLGDTATDFGAKVLDNTVSAILQLREHLNFEHVENAIEILNGARRIEFYGLGNSN IVAQDAHYKFFRFGIPTIAYGDLYMQAASAALLGKGDVIVAVSKSGRAPELLRVLDVAMQAGAKVIAITSSNTPLAKRATVALETDHIEMRESQLSMISRILHLLMIDILAVGVAIRRAS TNGELPEAVAQAKARASDDETADVLDWLSHGASPAAKDVARD

How is Recombinant Burkholderia cenocepacia Bifunctional protein glk typically supplied and what are the optimal storage conditions?

The protein is typically supplied as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . For optimal stability and activity retention, researchers should adhere to the following storage protocol:

• Store the lyophilized product at -20°C to -80°C upon receipt

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is standard) to prevent freeze-thaw damage

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• Store long-term aliquots at -20°C to -80°C

The protein's activity can be significantly compromised by repeated freeze-thaw cycles, so careful aliquoting after initial reconstitution is strongly recommended for maintaining consistent experimental results across multiple studies .

What is known about the functional domains and activities of Burkholderia cenocepacia Bifunctional protein glk?

While the search results provide limited information about the specific functional domains of this protein, the "bifunctional" designation suggests that the protein likely performs dual enzymatic or regulatory roles. Based on other GLK (GOLDEN2-LIKE) proteins studied in different contexts, these proteins often function as transcriptional regulators involved in developmental processes .

Experimental validation of the specific functions through enzymatic assays, substrate binding studies, and structural analysis would be necessary to fully characterize this protein's bifunctional nature.

What methodological approaches are most effective for studying protein-protein interactions involving Recombinant Burkholderia cenocepacia Bifunctional protein glk?

For investigating protein-protein interactions involving Recombinant Burkholderia cenocepacia Bifunctional protein glk, researchers should consider a multi-technique approach:

• Pull-down assays: Using the His-tag present on the recombinant protein for affinity purification can help identify binding partners. This technique has been successfully employed for other GLK proteins, as demonstrated in studies of GLK-HY5 interactions .

• Bimolecular luminescence complementation assays: This in vivo technique allows visualization of protein interactions in live cells. The protocol typically involves:

  • Fusing the protein of interest and potential interacting partners to complementary fragments of luciferase

  • Co-transfecting cells with both constructs

  • Measuring luminescence when protein interaction brings the luciferase fragments into proximity

• Co-immunoprecipitation: This technique can confirm in vivo interactions by:

  • Expressing tagged versions of the protein in an appropriate cell system

  • Precipitating with antibodies against the tag

  • Analyzing co-precipitated proteins by Western blot

• Electrophoretic Mobility Shift Assays (EMSA): If the protein has DNA-binding capabilities, EMSA can be used to study protein-DNA interactions by:

  • Generating Cy5-labeled DNA probes containing potential binding sites

  • Incubating purified protein with labeled probes in binding buffer

  • Resolving the reactions on a native polyacrylamide gel

  • Detecting shifted bands indicating protein-DNA complexes

These methodologies should be adapted based on the specific research questions and the cellular context in which the Burkholderia cenocepacia Bifunctional protein glk functions.

What are the challenges in expressing and purifying functionally active Recombinant Burkholderia cenocepacia Bifunctional protein glk?

Expressing and purifying functionally active Recombinant Burkholderia cenocepacia Bifunctional protein glk presents several challenges that researchers should address through careful optimization:

• Protein solubility: The large size (642 amino acids) of the full-length protein may lead to aggregation or inclusion body formation during expression. Strategies to improve solubility include:

  • Optimizing expression temperature (typically lowering to 16-20°C)

  • Using solubility-enhancing fusion tags beyond the His tag

  • Testing different E. coli strains specialized for difficult protein expression

  • Employing solubility enhancers in the growth media

• Maintaining native conformation: The bifunctional nature of the protein suggests complex folding requirements. To preserve native structure:

  • Include appropriate cofactors during purification if known

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Consider adding stabilizing agents like trehalose (already included in the commercial preparation at 6%)

• Activity preservation: The standard reconstitution and storage guidelines provided with commercial preparations should be strictly followed, with particular attention to:

  • Avoiding repeated freeze-thaw cycles

  • Maintaining appropriate glycerol concentration (5-50%) in storage buffers

  • Following recommended temperature conditions for short-term (4°C) and long-term (-20°C/-80°C) storage

• Purity assessment: While standard preparations achieve >90% purity by SDS-PAGE , researchers requiring higher purity for specific applications should consider:

  • Additional purification steps like size exclusion chromatography

  • Activity-based purification if functional assays are available

  • Mass spectrometry analysis to identify any remaining contaminants

How might Recombinant Burkholderia cenocepacia Bifunctional protein glk interact with host immune systems during infection?

While the search results don't provide direct evidence for the specific role of Burkholderia cenocepacia Bifunctional protein glk in host-pathogen interactions, we can draw some hypotheses based on related research:

Some GLK proteins in different contexts have been implicated in immune signaling. For instance, GLK-IKKβ signaling has been shown to induce dimerization and translocation in T cells, affecting IL-17A transcription through phosphorylation-mediated interactions . While this refers to a different GLK protein, it suggests that proteins in this family can have immunomodulatory effects.

For studying potential immune interactions of Recombinant Burkholderia cenocepacia Bifunctional protein glk, researchers should consider:

• In vitro immune cell stimulation assays:

  • Exposing macrophages, dendritic cells, or other immune cells to purified protein

  • Measuring cytokine production, cell activation markers, and pattern recognition receptor engagement

  • Comparing wild-type protein responses to mutated versions

• Structural analysis for molecular patterns:

  • Examining the protein for pathogen-associated molecular patterns (PAMPs)

  • Identifying regions that might interact with pattern recognition receptors

  • Using bioinformatics to predict immunogenic epitopes

• Comparative studies with clinical isolates:

  • Analyzing glk expression levels in virulent versus attenuated strains

  • Correlating expression with disease severity in cystic fibrosis patients infected with B. cenocepacia

  • Examining glk sequence variation across clinical isolates

Research in this direction could provide valuable insights into the role of this protein in Burkholderia cenocepacia pathogenesis, particularly in the context of pulmonary infections in immunocompromised patients.

What are the optimal reconstitution protocols for Recombinant Burkholderia cenocepacia Bifunctional protein glk to ensure maximum activity?

For optimal reconstitution of Recombinant Burkholderia cenocepacia Bifunctional protein glk, researchers should follow this detailed protocol:

• Pre-reconstitution preparation:

  • Briefly centrifuge the vial containing lyophilized protein to bring contents to the bottom

  • Allow the vial to reach room temperature before opening to prevent moisture condensation

• Reconstitution procedure:

  • Reconstitute in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL

  • Gently mix by inversion rather than vortexing to prevent protein denaturation

  • Allow the solution to sit at room temperature for 5-10 minutes to ensure complete dissolution

• Stabilization for storage:

  • Add glycerol to a final concentration of 50% (or between 5-50% as needed for downstream applications)

  • Mix thoroughly but gently

  • Prepare multiple small-volume aliquots to avoid repeated freeze-thaw cycles

• Storage recommendations:

  • Store working aliquots at 4°C for up to one week

  • Store long-term aliquots at -20°C/-80°C

  • Label tubes with reconstitution date, concentration, and buffer composition

• Activity verification:

  • If possible, perform a functional assay after reconstitution to confirm activity

  • Compare activity levels across different reconstitution methods if optimizing for specific applications

Following this protocol will help ensure that the reconstituted protein maintains its structural integrity and functional activity for reliable experimental results.

What experimental controls should be included when using Recombinant Burkholderia cenocepacia Bifunctional protein glk in functional studies?

When designing experiments with Recombinant Burkholderia cenocepacia Bifunctional protein glk, implementing appropriate controls is crucial for reliable and interpretable results:

• Negative controls:

  • Buffer-only controls that contain all components used in protein storage/reconstitution

  • Heat-denatured protein to demonstrate specificity of activity

  • Unrelated proteins of similar size and tag composition

• Positive controls:

  • Well-characterized proteins with similar functions, if available

  • Previously validated batches of the same protein

  • Native (non-recombinant) protein isolated from Burkholderia cenocepacia, if feasible

• Tag-specific controls:

  • Another His-tagged protein to control for tag-specific effects

  • The same protein with a different tag or no tag, if available

  • Tag-only constructs to isolate tag-mediated interactions

• Concentration controls:

  • Dose-response experiments to establish optimal protein concentrations

  • Titration series to determine the linear range of activity

  • Standardization to a known activity unit rather than protein mass

• Specificity controls for interaction studies:

  • Competitive binding experiments with unlabeled protein

  • Mutated versions of the protein targeting key functional residues

  • Domain-specific constructs to map interaction regions

• System-specific controls:

  • Cell-type specific controls when working in different host systems

  • Appropriate genetic knockout/knockdown controls to demonstrate specificity

  • Environmental condition controls (temperature, pH, ion concentrations)

Documenting and reporting these controls systematically will strengthen the validity of research findings and facilitate reproducibility across different laboratories.

How can researchers effectively compare different preparations or batches of Recombinant Burkholderia cenocepacia Bifunctional protein glk?

Consistent comparison between different preparations or batches of Recombinant Burkholderia cenocepacia Bifunctional protein glk requires standardized analytical methods:

• Physicochemical characterization:

  • SDS-PAGE for purity assessment and molecular weight confirmation

  • Western blotting with anti-His antibodies to verify tag presence and integrity

  • Mass spectrometry for precise molecular mass determination and sequence coverage

  • Circular dichroism to assess secondary structure composition

• Functional characterization:

  Assessment Method	Parameters to Measure	Acceptance Criteria
  Activity assay	Specific activity (units/mg)	≥80% of reference standard
  Binding affinity	K₁ for known substrates	Within 20% of reference value
  Thermal stability	Melting temperature (Tm)	±2°C of reference standard
  Aggregation profile	Dynamic light scattering	Monodisperse, PDI <0.2

• Standardization approaches:

  • Establish an internal reference standard from a well-characterized batch

  • Normalize all activity measurements to this standard

  • Develop and maintain a batch-to-batch comparison database

  • Consider including a standard curve with each new experiment

• Stability indicators:

  • Monitor activity retention over time under defined storage conditions

  • Assess freeze-thaw stability by measuring activity after multiple cycles

  • Evaluate thermal stress resistance at different temperatures

  • Test pH stability across the physiological range

• Documentation requirements:

  • Maintain detailed records of expression conditions for each batch

  • Document purification protocols with any deviations noted

  • Record storage history including temperature excursions

  • Create certificates of analysis for each preparation

By implementing these systematic comparison methods, researchers can minimize variability introduced by different protein preparations and ensure experimental reproducibility across studies.

What are the potential applications of Recombinant Burkholderia cenocepacia Bifunctional protein glk in studying bacterial pathogenesis?

Recombinant Burkholderia cenocepacia Bifunctional protein glk offers several valuable applications for investigating bacterial pathogenesis:

• Host-pathogen interaction studies:

  • The purified protein can be used to identify host cell receptors or targets

  • Protein-coated beads or surfaces can help study adhesion mechanisms

  • Labeled protein can track localization within host cells during infection

• Virulence factor characterization:

  • Comparing wild-type and mutant protein effects on host cells

  • Assessing the protein's role in biofilm formation

  • Determining if the protein contributes to antibiotic resistance mechanisms

• Immune response evaluation:

  • Measuring host immune cell activation in response to the protein

  • Assessing cytokine profiles induced by protein exposure

  • Determining if the protein has immunomodulatory properties that benefit bacterial survival

• Drug target validation:

  • Using the recombinant protein for high-throughput inhibitor screening

  • Structure-based drug design targeting specific protein domains

  • Evaluating compound effects on protein function in controlled in vitro systems

• Vaccine development research:

  • Assessing the protein as a potential immunogen

  • Evaluating antibody responses to various protein epitopes

  • Testing if antibodies against the protein offer protection in infection models

These applications could significantly advance our understanding of Burkholderia cenocepacia pathogenesis, particularly in the context of respiratory infections in immunocompromised patients and those with cystic fibrosis.

How might protein engineering approaches be applied to study structure-function relationships in Recombinant Burkholderia cenocepacia Bifunctional protein glk?

Protein engineering offers powerful approaches to dissect the structure-function relationships of Recombinant Burkholderia cenocepacia Bifunctional protein glk:

• Domain truncation studies:

  • Generate systematic truncation constructs to isolate functional domains

  • Express and characterize each domain independently

  • Reconstitute activity through domain complementation experiments

• Site-directed mutagenesis:

  • Target conserved residues identified through sequence alignment

  • Create alanine-scanning libraries across predicted active sites

  • Introduce mutations that alter charge, hydrophobicity, or structural features

• Fusion protein approaches:

  Fusion Partner	Purpose	Expected Outcome
  Fluorescent proteins	Real-time localization	Visualization of protein trafficking
  Split reporter systems	Protein-protein interaction detection	Signal upon interaction with partners
  Crystallization chaperones	Structure determination	Enhanced crystallization properties
  Ligand-binding domains	Controlled activation	Inducible protein function

• Directed evolution:

  • Develop selection systems for enhanced protein functions

  • Create libraries with random or targeted mutations

  • Select variants with desired properties (stability, activity, specificity)

  • Sequence selected variants to identify beneficial mutations

• Computational design and validation:

  • Use homology modeling to predict protein structure

  • Apply in silico mutagenesis to identify critical residues

  • Design stabilizing mutations based on computational predictions

  • Validate computational models through experimental testing

These approaches would yield valuable insights into how the bifunctional nature of the protein is encoded in its structure and could potentially lead to engineered variants with enhanced stability or novel functions for biotechnological applications.

What emerging technologies could advance our understanding of the role of Burkholderia cenocepacia Bifunctional protein glk in bacterial metabolism and pathogenesis?

Several cutting-edge technologies offer promising approaches to deepen our understanding of Burkholderia cenocepacia Bifunctional protein glk:

• Cryo-electron microscopy (Cryo-EM):

  • Determine high-resolution structure without crystallization

  • Visualize the protein in different functional states

  • Capture protein-protein or protein-substrate complexes

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map protein dynamics and conformational changes

  • Identify regions involved in ligand binding

  • Detect structural perturbations upon mutation

• CRISPR-Cas9 genome editing in Burkholderia:

  • Generate precise knockouts or mutations in the native gene

  • Create tagged versions for in situ localization

  • Develop conditional expression systems to study essentiality

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to measure conformational changes

  • Optical tweezers to study protein-protein interaction forces

  • Single-molecule tracking in live bacteria to monitor protein dynamics

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network analysis to position the protein in bacterial metabolic pathways

  • Flux balance analysis to predict metabolic consequences of protein dysfunction

• Advanced infection models:

  • Organ-on-a-chip systems to model complex host-pathogen interactions

  • 3D bioprinted tissue models for infection studies

  • Patient-derived cell models for personalized infection research

• Structural proteomics:

  • Crosslinking mass spectrometry to map interaction interfaces

  • Limited proteolysis to identify domain boundaries and flexible regions

  • Thermal proteome profiling to detect ligand-induced stabilization

These technologies, particularly when used in combination, could provide unprecedented insights into how Burkholderia cenocepacia Bifunctional protein glk functions within bacterial cells and during host infection, potentially revealing new therapeutic targets.

What strategies can researchers employ when facing low solubility or aggregation of Recombinant Burkholderia cenocepacia Bifunctional protein glk?

When encountering solubility or aggregation issues with Recombinant Burkholderia cenocepacia Bifunctional protein glk, researchers can implement these evidence-based strategies:

• Expression optimization:

  • Reduce expression temperature to 16-18°C

  • Use slower induction with lower IPTG concentrations (0.1-0.2 mM)

  • Test different E. coli strains (BL21, Rosetta, Arctic Express)

  • Consider co-expression with chaperones (GroEL/ES, DnaK/J)

• Buffer optimization:

  • Screen buffer compositions systematically

  • Test various pH conditions (typically pH 6.5-8.5)

  • Include stabilizing additives (glycerol, trehalose, arginine)

  • Add mild detergents (0.05% Tween-20 or Triton X-100)

• Solubilization approaches:

  Approach	Method	Considerations
  Fusion tags	MBP, SUMO, or TF tags	May affect protein function
  Refolding	Denaturation and controlled refolding	Often reduces yield and activity
  Inclusion body processing	Extraction under denaturing conditions	Requires optimization of refolding
  Domain-based expression	Express functional domains separately	May lose interdomain interactions

• Analytical techniques to guide optimization:

  • Use dynamic light scattering to monitor aggregation state

  • Apply differential scanning fluorimetry to identify stabilizing conditions

  • Employ size exclusion chromatography to quantify aggregation

  • Use analytical ultracentrifugation to characterize oligomeric states

• Storage and handling modifications:

  • Maintain protein at higher concentrations to prevent surface adsorption

  • Add carrier proteins (BSA) for very dilute solutions

  • Filter solutions through 0.22 μm filters before storage

  • Avoid freeze-thaw cycles by preparing single-use aliquots

Systematic documentation of conditions tested and their outcomes will help identify the optimal parameters for maintaining this challenging protein in a soluble, functional state.

How can researchers troubleshoot inconsistent activity of Recombinant Burkholderia cenocepacia Bifunctional protein glk across experiments?

Inconsistent activity of Recombinant Burkholderia cenocepacia Bifunctional protein glk across experiments can be methodically addressed through the following approaches:

• Standardization of protein handling:

  • Implement strict temperature control during all handling steps

  • Use consistent buffer compositions across experiments

  • Adopt single-use aliquots to eliminate freeze-thaw variation

  • Process all samples identically (mixing methods, incubation times)

• Activity assay optimization:

  • Determine the linear range of the activity assay

  • Establish reproducible positive controls for normalization

  • Optimize enzyme concentration and reaction time

  • Control for inhibitory or activating contaminants

• Systematic investigation of variables:

  • Create a decision tree of potential factors affecting activity

  • Test each variable independently while controlling others

  • Document all experimental conditions meticulously

  • Analyze patterns in activity variation across conditions

• Protein quality assessment:

  • Verify protein integrity before each experiment (SDS-PAGE)

  • Monitor protein stability during storage (activity time course)

  • Check for post-translational modifications or degradation (mass spectrometry)

  • Assess batch-to-batch variation through comparative analysis

• Environmental factor control:

  • Standardize laboratory temperature and humidity

  • Use calibrated pipettes and verified reagents

  • Control light exposure if the protein is photosensitive

  • Account for seasonal variations in water quality or reagents

• Statistical approaches:

  • Perform sufficient replicates (minimum n=3)

  • Apply appropriate statistical tests for significance

  • Establish acceptance criteria for experimental validity

  • Use control charts to track activity trends over time

By systematically addressing these potential sources of variability, researchers can significantly improve the reproducibility of experiments involving Recombinant Burkholderia cenocepacia Bifunctional protein glk.

What are the most promising research directions for understanding the role of Burkholderia cenocepacia Bifunctional protein glk in bacterial physiology and pathogenesis?

Based on current knowledge and technological capabilities, several high-priority research directions could significantly advance our understanding of Burkholderia cenocepacia Bifunctional protein glk:

• Structural biology:

  • Determine the three-dimensional structure through X-ray crystallography or cryo-EM

  • Identify the molecular basis for the protein's bifunctional nature

  • Map substrate binding sites and catalytic residues

• Functional genomics:

  • Generate and characterize glk knockout strains

  • Perform genome-wide synthetic lethal screens to identify genetic interactions

  • Use RNA-seq to identify transcriptional changes in glk mutants

  • Employ ChIP-seq if the protein has DNA-binding capabilities

• Metabolic role characterization:

  • Identify metabolic pathways affected by glk function

  • Determine if the protein plays a role in carbon metabolism

  • Investigate connections to stress responses or adaptation mechanisms

  • Assess the impact on bacterial growth under various conditions

• Pathogenesis studies:

  • Evaluate the contribution of glk to virulence in infection models

  • Determine if the protein affects antibiotic susceptibility

  • Investigate potential roles in biofilm formation or persistence

  • Assess interactions with host immune components

• Therapeutic target assessment:

  • Develop high-throughput screening methods for inhibitor discovery

  • Conduct fragment-based drug discovery if structural data becomes available

  • Evaluate the essentiality of the protein under infection-relevant conditions

  • Assess conservation across clinically relevant Burkholderia species