Recombinant Burkholderia pseudomallei Bifunctional protein glk (glk)

How should recombinant Burkholderia pseudomallei Bifunctional protein glk be stored for optimal stability?

For optimal stability, the recombinant Bifunctional protein glk should be stored at -20°C in its storage buffer (Tris-based buffer with 50% glycerol). For extended storage periods, -80°C is recommended to maintain protein integrity. Working aliquots can be maintained at 4°C for up to one week, but repeated freezing and thawing cycles should be avoided as they can lead to protein denaturation and loss of activity .

A recommended storage protocol includes:

• Upon receipt, briefly centrifuge the protein vial

• Prepare small working aliquots (10-20 μL) in sterile microcentrifuge tubes

• Store the majority of aliquots at -80°C for long-term preservation

• Keep 1-2 aliquots at -20°C for immediate use

• Maintain current working aliquot at 4°C for no more than 7 days

• Always thaw frozen aliquots on ice to minimize thermal stress

What is the genomic context of the glk gene in Burkholderia pseudomallei?

The glk gene is part of the complex genomic architecture of Burkholderia pseudomallei, which consists of two chromosomes of 4.07 and 3.17 megabase pairs. The genome displays significant functional partitioning between these chromosomes, with the larger one encoding core functions associated with central metabolism and cell growth, while the smaller chromosome carries more accessory functions related to adaptation and survival in different environments .

The genome of B. pseudomallei also features 16 genomic islands (GIs) that constitute approximately 6.1% of the total genome. These islands demonstrate variable presence across different isolates (both invasive and environmental soil samples) but are notably absent in the closely related species B. mallei . This genomic plasticity contributes to the pathogen's adaptability and virulence capabilities.

The functional partitioning observed in the B. pseudomallei genome is comparable to that seen in Ralstonia solanacearum, which also possesses a bipartite genome structure with a 3.7 Mb chromosome and a 2.0 Mb megaplasmid .

What are the most effective methods for recombinant expression of the Bifunctional protein glk?

Effective recombinant expression of Bifunctional protein glk from B. pseudomallei can be achieved through several approaches, with promoter replacement strategies being particularly successful. The following methodology has demonstrated high efficiency:

• Design a promoter-replacement strategy using the rhamnose-inducible promoter PRhaB

• Create recombination cassettes targeting promoters upstream of biosynthetic-rich operons

• Ligate these cassettes into an allele replacement vector (e.g., pEXKm5)

• Transform the vector into E. coli S17-1 for conjugation with B. pseudomallei

• Select transconjugants using appropriate antibiotics (kanamycin at 1000 μg/mL)

For transformation specifically involving the attenuated B. pseudomallei strain Bp82 (derived from Bp1026b through deletion of the ΔpurM gene), the following protocol is recommended:

• Supplement all media with adenine (80 μg/mL) and thymidine (5 μg/mL)

• Construct promoter exchange cassettes containing the rhamnose-inducible promoter PRhaB and the dhfr trimethoprim resistance gene

• PCR amplify the ~4.8 kb promoter exchange cassette

• Process for blunt-ended phosphorylation and ligate into vector pEXKm5

• Transport into E. coli S17-1 and conjugate with Bp82

• Select on LB agar with kanamycin (1000 μg/mL) and trimethoprim (100 μg/mL)

• Add gentamycin (20 μg/mL) to counterselect against E. coli

How can enzymatic activity of the glucokinase domain be specifically measured?

The glucokinase domain (EC 2.7.1.2) of the Bifunctional protein glk catalyzes the phosphorylation of glucose to glucose-6-phosphate using ATP as a phosphate donor. To specifically measure this activity, researchers can employ the following methodology:

• Spectrophotometric coupled assay: Measure the production of glucose-6-phosphate by coupling it to glucose-6-phosphate dehydrogenase reaction, which produces NADPH that can be detected at 340 nm.

Reaction components:

• Recombinant Bifunctional protein glk (10-50 μg/mL)

• D-glucose (1-10 mM)

• ATP (1-5 mM)

• MgCl₂ (5-10 mM)

• NADP⁺ (0.5 mM)

• Glucose-6-phosphate dehydrogenase (2-5 U/mL)

• Buffer: 50 mM Tris-HCl, pH 7.5

Procedure:

• Prepare reaction mixture excluding the recombinant protein

• Equilibrate at 37°C for 5 minutes

• Add recombinant protein to initiate the reaction

• Monitor increase in absorbance at 340 nm continuously

• Calculate activity using the molar extinction coefficient of NADPH (6,220 M⁻¹cm⁻¹)

• Radiometric assay: Using [γ-³²P]ATP to monitor the transfer of radioactive phosphate to glucose.

• Enzyme-coupled fluorescence assay: Coupling the reaction to NADPH production and measuring fluorescence (excitation 340 nm, emission 460 nm).

What protocols are most suitable for purifying recombinant Bifunctional protein glk?

Purification of recombinant Bifunctional protein glk requires a multi-step approach that preserves both enzymatic and DNA-binding activities. The following protocol is recommended:

Chromatography-based purification protocol:

• Cell lysis:

  • Resuspend cells in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 5% glycerol, protease inhibitor cocktail)

  • Lyse cells using sonication or mechanical disruption

  • Clarify lysate by centrifugation at 20,000 × g for 30 minutes at 4°C

• Immobilized Metal Affinity Chromatography (IMAC):

  • Load clarified lysate onto a Ni-NTA column pre-equilibrated with binding buffer

  • Wash with increasing concentrations of imidazole (20-50 mM)

  • Elute protein with elution buffer containing 250-300 mM imidazole

• Ion Exchange Chromatography:

  • Dialyze IMAC fractions against low-salt buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl)

  • Load onto Q-Sepharose column

  • Elute with linear gradient of NaCl (50-500 mM)

• Size Exclusion Chromatography:

  • Concentrate pooled fractions using centrifugal filter units

  • Apply to Superdex 200 column equilibrated with storage buffer

  • Collect fractions containing purified protein

• Final formulation:

  • Buffer exchange into storage buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, 50% glycerol)

  • Filter-sterilize through 0.22 μm filter

  • Store at -20°C or -80°C in aliquots

Typical yield: 5-10 mg of purified protein per liter of bacterial culture with >95% purity as assessed by SDS-PAGE.

How does the dual functionality of glk contribute to B. pseudomallei pathogenesis?

The bifunctional nature of the glk protein potentially contributes to B. pseudomallei pathogenesis through multiple mechanisms:

• Metabolic adaptation: The glucokinase domain enables efficient glucose utilization in various host environments, supporting bacterial growth during infection. This metabolic flexibility is particularly important as B. pseudomallei transitions between extracellular and intracellular lifestyles during infection.

• Transcriptional regulation: The HTH-type transcriptional regulator domain likely controls expression of virulence factors or metabolic genes in response to environmental cues within the host. This regulatory capacity may enable rapid adaptation to changing conditions during the infection process.

• Contribution to genomic plasticity: As part of B. pseudomallei's complex genome, the glk gene may participate in the genomic diversification that characterizes this pathogen. The genome of B. pseudomallei contains 16 genomic islands that make up 6.1% of the total genome, conferring genetic diversity that enhances virulence and environmental adaptation .

• Integration with bacterial metabolism: The bifunctional nature of glk allows for coordinated regulation of glucose metabolism and gene expression, potentially creating regulatory circuits that respond to metabolic states during infection.

A comprehensive understanding of these mechanisms requires further investigation using knockout mutants and complementation studies to directly assess the contribution of each domain to virulence in animal models of melioidosis.

What approaches can be used to investigate the DNA-binding specificity of the HTH domain?

The HTH (helix-turn-helix) domain of the Bifunctional protein glk is predicted to function as a transcriptional regulator. To investigate its DNA-binding specificity, researchers can employ several complementary approaches:

• Chromatin Immunoprecipitation sequencing (ChIP-seq):

  • Cross-link protein-DNA complexes in vivo

  • Immunoprecipitate using antibodies against the recombinant protein

  • Sequence bound DNA fragments to identify genome-wide binding sites

  • Analyze sequences for conserved motifs using algorithms like MEME or HOMER

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified recombinant protein with labeled DNA fragments

  • Analyze protein-DNA complexes by native gel electrophoresis

  • Perform competition assays with unlabeled DNA to determine specificity

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX):

  • Incubate protein with a randomized DNA oligonucleotide library

  • Isolate bound sequences and amplify by PCR

  • Repeat selection process through multiple rounds

  • Sequence enriched oligonucleotides to identify consensus binding motifs

• DNA footprinting:

  • Incubate labeled DNA with purified protein

  • Treat with DNase I or chemical cleavage agents

  • Analyze protected regions by sequencing gel electrophoresis

• Reporter gene assays:

  • Clone potential target promoters upstream of reporter genes

  • Co-express the HTH domain or full-length protein

  • Measure reporter activity to assess transcriptional effects

How can recombinant Bifunctional protein glk be used in vaccine development against melioidosis?

Recombinant Bifunctional protein glk holds potential as a vaccine candidate against melioidosis due to several characteristics:

• Conserved nature: As a metabolic enzyme with regulatory functions, glk is likely well-conserved across B. pseudomallei strains, potentially providing broad protection.

• Dual functionality: The protein's bifunctional nature may elicit immune responses against multiple epitopes, potentially enhancing protective efficacy.

• Potential immunogenicity: Bacterial metabolic enzymes often display good immunogenicity when used as vaccine antigens.

A systematic approach to evaluating glk as a vaccine candidate would include:

Phase 1: Immunogenicity Assessment

• Evaluate humoral immune responses by measuring antibody titers in animal models

• Characterize cell-mediated immunity through T-cell proliferation assays

• Identify immunodominant epitopes through epitope mapping

Phase 2: Protection Studies

• Challenge immunized animals with virulent B. pseudomallei

• Measure survival rates, bacterial burden, and inflammatory markers

• Compare with established vaccine candidates

Phase 3: Delivery System Optimization

• Evaluate different adjuvant formulations

• Test prime-boost strategies combining protein antigen with DNA vaccines

• Develop nanoparticle or liposome-based delivery systems

What controls should be included when assessing glk enzymatic activity in experimental settings?

When assessing the enzymatic activity of Bifunctional protein glk, the following controls should be included to ensure experimental validity:

• Negative controls:

  • Reaction mixture without the recombinant protein

  • Heat-inactivated protein (95°C for 10 minutes)

  • Reaction without substrate (glucose)

  • Reaction without cofactor (ATP)

• Positive controls:

  • Commercial glucokinase with known activity

  • Previously characterized batch of the recombinant protein

• Specificity controls:

  • Alternate hexose substrates (mannose, fructose) to assess substrate specificity

  • Alternative phosphate donors (GTP, UTP) to assess nucleotide specificity

• Inhibition controls:

  • Known glucokinase inhibitors (N-acetylglucosamine, mannoheptulose)

  • Product inhibition assessment (glucose-6-phosphate)

• Buffer and condition controls:

  • pH range (6.0-9.0) to determine pH optimum

  • Temperature range (25-45°C) to determine temperature optimum

  • Metal ion dependency (Mg²⁺, Mn²⁺, Ca²⁺)

A comprehensive enzymatic characterization should include:

How can protein-protein interactions of Bifunctional protein glk be identified and characterized?

Identifying and characterizing protein-protein interactions (PPIs) of Bifunctional protein glk requires a multi-faceted approach:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged recombinant protein in B. pseudomallei or heterologous system

  • Perform pull-down using affinity resin

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions by reciprocal pull-downs

• Yeast two-hybrid (Y2H) screening:

  • Clone glk as bait in Y2H vector

  • Screen against B. pseudomallei genomic DNA library

  • Validate positive interactions by secondary assays

• Biolayer interferometry (BLI) or Surface Plasmon Resonance (SPR):

  • Immobilize purified recombinant protein on sensor

  • Flow potential interacting proteins over sensor

  • Measure association and dissociation kinetics

  • Calculate binding affinities (KD values)

• Protein cross-linking combined with mass spectrometry:

  • Treat B. pseudomallei lysates with protein cross-linkers

  • Purify glk complexes

  • Identify cross-linked peptides by mass spectrometry

  • Map interaction interfaces

• Co-immunoprecipitation from native conditions:

  • Generate specific antibodies against glk

  • Immunoprecipitate from B. pseudomallei lysates

  • Identify co-precipitating proteins by Western blot or mass spectrometry

Characterization of identified interactions should include:

• Determination of binding affinities

• Mapping of interaction domains

• Functional consequences of the interaction

• Regulation of interactions by metabolites or environmental conditions

What are the challenges in developing specific inhibitors targeting the Bifunctional protein glk?

Developing specific inhibitors for Bifunctional protein glk presents several unique challenges due to its bifunctional nature and biological context:

• Dual functionality: Inhibitors may need to target either the glucokinase domain, the HTH domain, or both, depending on the desired effect. This complexity requires careful structure-activity relationship studies.

• Selectivity concerns: Ensuring selectivity against human hexokinases is crucial to avoid off-target effects. The prokaryotic glucokinase domain differs structurally from human hexokinases, but identifying selective inhibitory scaffolds remains challenging.

• Structural considerations:

  • Limited structural information on B. pseudomallei glk

  • Potential conformational changes between active and inactive states

  • Interdomain interactions affecting inhibitor binding

• Drug delivery challenges:

  • Penetration of the B. pseudomallei cell envelope

  • Efflux pump-mediated resistance

  • Intracellular delivery for targeting bacteria within host cells

• Resistance development: B. pseudomallei's genomic plasticity facilitates rapid adaptation, potentially leading to resistance through mutations or gene acquisition .

A systematic inhibitor development pipeline should include:

How can recombinant Bifunctional protein glk contribute to diagnostic development for melioidosis?

Recombinant Bifunctional protein glk offers several advantages for developing improved diagnostics for melioidosis:

• Serological diagnostics:

  • Development of ELISA-based tests using recombinant glk as capture antigen

  • Lateral flow immunoassays for rapid point-of-care testing

  • Multiplexed assays combining glk with other B. pseudomallei antigens

• Molecular diagnostics:

  • PCR primers targeting the glk gene sequence

  • LAMP (Loop-mediated isothermal amplification) assays for resource-limited settings

  • Digital PCR for quantitative detection from clinical samples

• Antigen detection:

  • Monoclonal antibodies against glk for direct antigen detection

  • Mass spectrometry-based identification from clinical isolates

The unique bifunctional nature of the protein provides multiple epitopes for antibody recognition, potentially improving assay sensitivity and specificity. Considering that melioidosis causes approximately 20% of community-acquired septicemias in northeastern Thailand with a 50% mortality rate, improved diagnostics could significantly impact patient outcomes .

A development pipeline for glk-based diagnostics would include:

What emerging technologies could advance research on Bifunctional protein glk?

Several emerging technologies hold promise for advancing research on Bifunctional protein glk:

• CRISPR-Cas9 genome editing:

  • Precise modification of glk in B. pseudomallei

  • Domain-specific mutations to dissect function

  • CRISPRi for conditional repression to study essentiality

• Single-cell technologies:

  • Single-cell RNA-seq to study glk expression heterogeneity

  • Single-cell proteomics to assess protein levels

  • Single-bacterium imaging to track localization

• Cryo-electron microscopy (Cryo-EM):

  • High-resolution structural determination

  • Visualization of conformational states

  • Complex formation with interaction partners

• Microfluidics applications:

  • Encapsulation of single bacteria for phenotypic studies

  • Droplet-based enzymatic assays

  • Organ-on-chip models for infection studies

• Artificial intelligence approaches:

  • Protein structure prediction using AlphaFold2

  • Molecular dynamics simulations to study protein motion

  • Virtual screening for novel inhibitors

• Synthetic biology tools:

  • Designer circuits incorporating glk regulatory elements

  • Biosensors based on glk activity

  • Cell-free expression systems for functional studies

How does the research on Bifunctional protein glk contribute to understanding bacterial evolution and adaptation?

Research on Bifunctional protein glk provides valuable insights into bacterial evolution and adaptation, particularly regarding:

• Bifunctional protein evolution:

  • The glk protein represents an interesting case of domain fusion, combining metabolic and regulatory functions

  • This fusion may provide selective advantages through coordinated regulation

  • Comparative genomics can reveal the evolutionary history of this arrangement

• Genomic plasticity and horizontal gene transfer:

  • B. pseudomallei's genome demonstrates significant plasticity with 16 genomic islands comprising 6.1% of the genome

  • These islands show variable presence across different isolates but are absent in the related B. mallei

  • This variability contributes to B. pseudomallei's genetic diversity and adaptability

• Bipartite genome organization:

  • The bifunctional nature of glk mirrors the bipartite organization of the B. pseudomallei genome

  • The large chromosome (4.07 Mb) encodes core metabolic functions while the small chromosome (3.17 Mb) carries accessory functions for adaptation

  • This functional partitioning suggests distinct evolutionary origins for the two chromosomes

• Comparative genomics insights:

  • Orthologous matches to B. pseudomallei proteins are found in Ralstonia solanacearum (2,535 of 5,855 proteins)

  • R. solanacearum also has a bipartite genome structure (3.7 Mb chromosome + 2.0 Mb megaplasmid)

  • Chromosome 2 appears more divergent, containing a smaller percentage of orthologous genes

• Metabolic adaptation mechanisms:

  • The glucokinase domain represents a key entry point for carbon metabolism

  • Coupling with transcriptional regulation allows for responsive metabolic control

  • This arrangement may facilitate adaptation to diverse environmental niches

Understanding these evolutionary aspects provides broader context for biothreat research and pathogenesis studies, as B. pseudomallei is recognized as a biothreat agent and the causative agent of melioidosis, which accounts for 20% of community-acquired septicemias in northeastern Thailand .