Recombinant Bacillus thuringiensis subsp. konkukian UPF0754 membrane protein BT9727_0767 (BT9727_0767)

What expression systems are effective for producing recombinant BT9727_0767?

For recombinant production of BT9727_0767, E. coli expression systems have been successfully employed. The methodology typically involves:

• Gene synthesis or amplification of the BT9727_0767 coding sequence

• Cloning into a suitable expression vector containing a His-tag sequence

• Transformation into an E. coli expression strain

• Induction of protein expression under optimized conditions

• Purification using immobilized metal affinity chromatography (IMAC)

This approach has demonstrated reliable expression with purity greater than 90% as determined by SDS-PAGE analysis. When designing your expression system, consider codon optimization for E. coli if using synthetic genes to enhance expression levels.

What storage conditions maintain the stability of recombinant BT9727_0767?

Optimal storage conditions for recombinant BT9727_0767 include:

For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL. After reconstitution, add glycerol to a final concentration of 50% for long-term storage. Avoid repeated freeze-thaw cycles, as they can significantly reduce protein activity and stability.

What reconstitution protocol is recommended for optimal BT9727_0767 stability?

The recommended reconstitution protocol for BT9727_0767 involves several critical steps to ensure maximum stability and activity:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the lyophilized protein 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)

• Aliquot the solution 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

This protocol maintains protein in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps preserve structural integrity during freeze-thaw cycles. When working with membrane proteins like BT9727_0767, maintaining proper buffer conditions is particularly important for preventing aggregation.

How can researchers effectively solubilize and maintain the native conformation of BT9727_0767?

As a membrane protein, BT9727_0767 presents distinct challenges for solubilization while maintaining its native conformation. An effective methodological approach includes:

• Initial solubilization with mild detergents such as n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG)

• Determination of critical micelle concentration (CMC) for the selected detergent

• Gradual detergent removal using detergent-absorbing beads if reconstitution into lipid bilayers is desired

• Buffer optimization to include stabilizing agents (glycerol, trehalose)

• Temperature control during solubilization (typically 4°C)

For structural studies, consider screening multiple detergents and buffer conditions in parallel. Membrane proteins often require specific lipid environments to maintain their native conformation and function.

What approaches can be used to determine the membrane topology of BT9727_0767?

Determining the membrane topology of BT9727_0767 requires a multi-faceted experimental approach:

• Computational prediction: Use algorithms such as TMHMM, HMMTOP, or Phobius to predict transmembrane domains based on the amino acid sequence.

• Protease accessibility assays: Expose membrane vesicles containing BT9727_0767 to proteases. Regions accessible to proteolytic cleavage are likely exposed on the surface, while protected regions are embedded in the membrane.

• Cysteine scanning mutagenesis: Systematically replace individual amino acids with cysteine and then probe accessibility with membrane-permeable and membrane-impermeable sulfhydryl reagents.

• Epitope insertion: Insert small epitope tags at various positions and determine their accessibility via immunodetection.

• Fluorescence techniques: Use environment-sensitive fluorescent probes attached at specific positions to determine membrane insertion.

Based on the available sequence data, the C-terminal region (LLGGMIGIVQGLLLLFLK) shows high hydrophobicity characteristic of transmembrane domains, suggesting at least one membrane-spanning segment.

What structural biology techniques are most appropriate for studying BT9727_0767?

Multiple structural biology techniques can be applied to BT9727_0767, each with specific advantages:

For membrane proteins like BT9727_0767, cryo-EM has recently emerged as particularly valuable due to advances in direct detection cameras and image processing algorithms that can handle the challenges of membrane protein complexes embedded in detergent micelles or nanodiscs.

What is known about the biological function of BT9727_0767 and related UPF0754 proteins?

The UPF0754 membrane protein family, to which BT9727_0767 belongs, remains functionally uncharacterized (UPF stands for Uncharacterized Protein Family). Based on comparative genomics:

• UPF0754 proteins are conserved across various Bacillus species, suggesting important physiological functions

• The presence in Bacillus thuringiensis, an insecticidal bacterium, suggests possible roles in pathogenesis or environmental adaptation

• Related proteins exist in Bacillus cereus (BCE_0952, BCAH187_A1042, BCB4264_A0915) and Bacillus anthracis (BAMEG_3697), implying conservation across the Bacillus cereus group

To determine function, consider comparative functional genomics approaches by:

• Analyzing gene neighborhood conservation

• Examining co-expression patterns with characterized genes

• Performing knockout/knockdown studies followed by phenotypic analysis

• Conducting comparative transcriptomics under various stress conditions

Gene deletion studies in model Bacillus species could provide insights into the physiological role of this protein family.

How can researchers investigate potential protein-protein interactions of BT9727_0767?

To investigate protein-protein interactions involving BT9727_0767, several complementary approaches can be employed:

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

  • Express His-tagged BT9727_0767 in Bacillus thuringiensis

  • Perform crosslinking to stabilize transient interactions

  • Purify using nickel affinity chromatography

  • Identify co-purifying proteins by mass spectrometry

• Bacterial two-hybrid system:

  • Adapt yeast two-hybrid approach for bacterial membrane proteins

  • Screen against genomic libraries of B. thuringiensis

• Co-immunoprecipitation with specific antibodies:

  • Generate antibodies against BT9727_0767

  • Perform pulldown experiments from membrane fractions

• Proximity-dependent biotin labeling (BioID):

  • Fuse BT9727_0767 to a biotin ligase

  • Identify biotinylated neighbor proteins

• Membrane-specific crosslinking approaches:

  • Use membrane-permeable crosslinkers

  • Identify complexes by Western blotting and mass spectrometry

When designing these experiments, consider the importance of maintaining the native membrane environment to preserve physiologically relevant interactions. The search results do not indicate known interaction partners, suggesting this is an open area for investigation.

How does BT9727_0767 compare to UPF0754 proteins in other Bacillus species?

A comparative analysis of BT9727_0767 with UPF0754 proteins from other Bacillus species reveals evolutionary relationships and potential functional conservation:

This high sequence conservation across pathogenic and non-pathogenic Bacillus species suggests a fundamental role in bacterial physiology rather than a specific role in pathogenesis. The conservation pattern follows the established phylogenetic relationships within the Bacillus genus.

Sequence alignment and structural prediction tools can identify conserved domains and motifs that may indicate functional regions. Additionally, synteny analysis (examining the conservation of gene neighborhoods) can provide functional insights based on genomic context.

What experimental approaches can differentiate between the roles of various UPF0754 family members?

To differentiate between the roles of UPF0754 family members across different Bacillus species, consider these methodological approaches:

• Heterologous complementation studies:

  • Generate gene knockouts in model Bacillus species

  • Attempt to rescue phenotypes with UPF0754 genes from different species

  • Evaluate functional conservation/divergence

• Domain swapping experiments:

  • Create chimeric proteins combining domains from different UPF0754 proteins

  • Test functionality to identify species-specific functional domains

• Comparative transcriptomics:

  • Compare expression patterns of UPF0754 genes across species under identical conditions

  • Identify conserved and divergent regulatory patterns

• Differential interactome analysis:

  • Perform parallel protein-protein interaction studies for UPF0754 proteins from different species

  • Identify common and species-specific interaction partners

• Comparative phenotypic profiling:

  • Create parallel gene deletions across multiple Bacillus species

  • Compare phenotypic consequences under standardized conditions

These approaches can reveal whether UPF0754 proteins perform identical functions across species or have evolved species-specific roles, potentially related to niche adaptation or pathogenicity.

What strategies can overcome challenges in expressing and purifying sufficient quantities of BT9727_0767?

Membrane proteins like BT9727_0767 present unique challenges for expression and purification. Advanced strategies to overcome these include:

• Alternative expression hosts:

  • Beyond E. coli, consider Bacillus subtilis (closer native environment)

  • Insect cell expression (baculovirus system) for complex membrane proteins

  • Cell-free expression systems with supplied lipids/detergents

• Fusion protein approaches:

  • N-terminal fusions with highly soluble partners (MBP, SUMO, Trx)

  • GFP fusions for rapid assessment of folding and membrane integration

• Codon optimization and expression condition screening:

  • Design synthetic genes with optimized codon usage

  • Systematic screening of induction temperature, inducer concentration, and duration

• Specialized solubilization and purification:

  • Detergent screening matrix (12-24 different detergents)

  • Lipid-detergent mixed micelles

  • Nanodiscs or styrene maleic acid lipid particles (SMALPs) for native-like environment

• Scale-up strategies:

  • High-density fermentation

  • Membrane fractionation prior to solubilization

  • Automated purification systems

The available data confirms successful expression in E. coli with His-tag purification, but alternative approaches may be necessary depending on downstream applications and required protein quantities.

How can site-directed mutagenesis be applied to study structure-function relationships in BT9727_0767?

Site-directed mutagenesis offers a powerful approach to investigate structure-function relationships in BT9727_0767:

• Target selection strategy:

  • Conserved residues across UPF0754 family (likely functional importance)

  • Predicted transmembrane residues (role in membrane integration)

  • Charged residues in transmembrane regions (often functionally critical)

  • Predicted ligand-binding pockets

• Mutagenesis approach:

  • Alanine scanning of conserved regions

  • Conservative vs. non-conservative substitutions

  • Introduction of reporter residues (cysteine, tryptophan)

  • Creation of chimeric proteins with related UPF0754 members

• Functional analysis of mutants:

  • Membrane localization assessment

  • Protein stability and folding analysis

  • Interaction partner binding studies

  • In vivo complementation of knockout phenotypes

• Structural impact assessment:

  • Circular dichroism spectroscopy to assess secondary structure changes

  • Thermal stability measurements

  • Limited proteolysis patterns

The amino acid sequence provided in the search results can serve as the template for designing mutagenesis experiments, with particular attention to the hydrophobic regions likely involved in membrane interactions.

How might BT9727_0767 contribute to understanding bacterial membrane biology?

The study of BT9727_0767 can advance our understanding of bacterial membrane biology in several key ways:

• Membrane protein evolution: Comparative analysis across Bacillus species can illuminate how membrane proteins evolve while maintaining structural integrity in the hydrophobic membrane environment.

• Protein sorting and membrane insertion: As a membrane protein with predicted transmembrane segments, BT9727_0767 can serve as a model to study how Gram-positive bacteria target and insert proteins into membranes.

• Function of uncharacterized membrane proteins: The UPF0754 family represents one of many uncharacterized membrane protein families. Methodologies developed for BT9727_0767 characterization can be applied to other UPF proteins.

• Membrane organization in Bacillus species: Studies on BT9727_0767 localization can contribute to understanding membrane microdomains and protein clustering in bacterial membranes.

• Bacterial adaptation mechanisms: Comparing expression and function across different growth conditions may reveal roles in stress response or environmental adaptation.

Future research should consider integrating advanced imaging techniques like super-resolution microscopy to visualize the spatial organization of BT9727_0767 in the bacterial membrane.

What emerging technologies might advance the structural and functional characterization of BT9727_0767?

Several emerging technologies hold promise for advancing our understanding of BT9727_0767:

• Cryo-electron tomography:

  • Direct visualization of BT9727_0767 in its native membrane environment

  • No need for protein crystallization

  • Potential for in situ structural determination

• AlphaFold and deep learning structure prediction:

  • Accurate prediction of membrane protein structures

  • Generation of testable structural hypotheses

  • Guide for rational mutagenesis studies

• Single-molecule FRET:

  • Dynamic structural information on conformational changes

  • Real-time monitoring of protein-protein interactions

  • Information on potential ligand-induced conformational changes

• Native mass spectrometry for membrane proteins:

  • Determination of intact protein complexes

  • Analysis of lipid-protein interactions

  • Identification of potential small molecule binding

• Genome-wide interaction screens using CRISPR-Cas9:

  • Systematic identification of genetic interactions

  • Discovery of synthetic lethal partners

  • Pathway assignment through genetic relationships

• High-throughput automated crystallization pipelines:

  • Systematic screening of thousands of crystallization conditions

  • Specialized for membrane proteins with lipidic cubic phase methods

  • Miniaturized setups requiring less protein

These technologies can overcome traditional barriers to membrane protein research, potentially accelerating the structural and functional characterization of BT9727_0767 and related proteins.