Recombinant Shewanella halifaxensis Protein CrcB homolog (crcB)

What is the Recombinant Shewanella halifaxensis Protein CrcB homolog?

The Recombinant Shewanella halifaxensis Protein CrcB homolog (crcB) is a recombinant protein derived from the Shewanella halifaxensis strain HAW-EB4. This protein is characterized by its UniProt accession number B0TUP5 and is encoded by the gene designated as crcB (ordered locus name: Shal_2214). The protein consists of 124 amino acids in its expression region and is primarily associated with membrane functions in the bacterial cell . Scientifically, CrcB homologs are known to function in various cellular processes, often related to ion transport and membrane integrity. Understanding this protein requires contextualizing it within the broader family of CrcB homologs, which are evolutionarily conserved across diverse bacterial species.

What are the optimal storage conditions for Recombinant Shewanella halifaxensis Protein CrcB homolog?

For optimal preservation of protein integrity and activity, the Recombinant Shewanella halifaxensis Protein CrcB homolog should be stored in a Tris-based buffer with 50% glycerol. The primary storage temperature should be -20°C, while for extended storage periods, conservation at -80°C is recommended . When working with the protein, it is advisable to create working aliquots that can be stored at 4°C for up to one week to avoid repeated freeze-thaw cycles that can compromise protein stability . To methodologically verify the integrity of stored protein samples, researchers should consider implementing quality control measures such as SDS-PAGE analysis before experimental use to confirm protein integrity after storage periods.

What is the amino acid sequence of the Shewanella halifaxensis CrcB homolog protein?

The complete amino acid sequence of the Shewanella halifaxensis CrcB homolog protein is:
MNNVLFVALGGSIIGAVLRYLISLLMLQVFGSGFPFGTLVVNILGSFLMGVIFALG QVSELSPEIKAFIGVGMLGALTTFSTFSNESLLLMQEGELVKAVLNVVVNVGVCIF VVYLGQQLVFSRF

This sequence information is essential for researchers planning protein structure analyses, epitope mapping, or designing site-directed mutagenesis experiments. The protein sequence can be used for in silico structural predictions, phylogenetic analyses to identify conserved domains, or designing custom antibodies for experimental applications. Researchers should note that there may be small variations in commercially produced recombinant proteins depending on the expression system and purification methodology.

What experimental techniques are recommended for studying Shewanella halifaxensis proteins?

Based on established protocols for Shewanella species proteins, recommended experimental techniques include:

• Protein isolation and purification: Immunoprecipitation using GFP-Trap_MA beads has been successfully applied to Shewanella proteins, allowing specific isolation of tagged protein variants from cell lysates .

• Protein expression verification: Immunoblot analysis using polyclonal antibodies against specific tags (e.g., FLAG-tag) with chemiluminescent detection systems like SuperSignal West Pico Chemiluminescent Substrate has proven effective .

• Protein-protein interaction studies: Bacterial two-hybrid system (BATCH) approaches have been successfully implemented for Shewanella proteins to detect in vivo protein-protein interactions .

• Microscopy techniques: Fluorescence microscopy using agarose pad immobilization has been employed for visualizing tagged proteins in Shewanella species .

These methodologies provide researchers with a robust foundation for designing experiments to study the localization, interactions, and functions of the CrcB homolog protein.

What are the challenges in expressing Recombinant Shewanella halifaxensis Protein CrcB homolog in heterologous systems?

Expression of Recombinant Shewanella halifaxensis Protein CrcB homolog in heterologous systems presents several research challenges. A significant factor affecting expression success is the accessibility of translation initiation sites. Recent comprehensive analysis of recombinant protein production experiments has demonstrated that the mRNA base-unpairing across the Boltzmann's ensemble (modeled as accessibility) significantly outperforms alternative features in predicting expression success . For membrane proteins like CrcB homolog, additional considerations include:

• Translation initiation efficiency: The local features of mRNA, particularly around the -24:24 and -30:30 regions relative to the start codon, have been shown to be crucial predictors of expression success. The local G+C contents in these regions correlate with opening energy and Minimum Free Energy (MFE) respectively, though the accessibility measure itself is a more reliable predictor .

• Protein folding and membrane integration: As a membrane protein, CrcB homolog requires proper folding and insertion into membranes, which can be challenging in heterologous expression systems.

• Host-specific codon usage: While general codon adaptation index (CAI) shows some correlation with expression success, the impact appears secondary to translation initiation accessibility .

Researchers should consider optimizing the mRNA sequence around the initiation region rather than focusing exclusively on codon optimization of the entire coding sequence.

How can researchers implement genome editing to study CrcB homolog function in Shewanella halifaxensis?

To conduct functional studies of the CrcB homolog in Shewanella halifaxensis, researchers can implement CRISPR/Cas9-based genome editing systems that have been successfully demonstrated in Shewanella species. A highly efficient approach involves using single-stranded DNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection . The methodological workflow involves:

• Two-plasmid system setup: Utilize a sgRNA targeting vector and an editing vector harboring both Cas9 and phage recombinase W3 Beta .

• Design of targeting components: Create specific sgRNAs targeting the crcB gene locus.

• Design of recombination template: Synthesize single-stranded DNA as substrates for homologous recombination that incorporate desired genetic changes (mismatches, deletions, or small insertions) .

• Transformation and selection: Transform both plasmids into Shewanella halifaxensis and select transformants.

This system offers an average efficiency of >90% among transformed cells, compared to approximately 5% by recombineering alone . For verification of successful genomic modifications, researchers should implement colony PCR screening followed by sequencing of the target locus to confirm the precision of the introduced genetic changes.

What protein purification methods are most effective for Recombinant Shewanella halifaxensis Protein CrcB homolog?

For efficient purification of Recombinant Shewanella halifaxensis Protein CrcB homolog, a multistep approach is recommended based on successful purification protocols for other Shewanella proteins. The methodology should account for the membrane-associated nature of the CrcB homolog:

• Cell lysis optimization: Sonication in a buffer containing 50 mM Tris-HCl, 250 mM NaCl, 25 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 0.01% sodium azide, 5% glycerol, and an appropriate concentration of imidazole (typically 40 mM for initial binding) has proven effective for Shewanella proteins .

• Immobilized metal affinity chromatography (IMAC): For His-tagged versions of the protein, use of a 5-ml HisTrap column equilibrated with the lysis buffer, followed by washing and elution with increased imidazole concentration (300 mM) has shown good results with Shewanella proteins .

• Tag removal considerations: If the tag interferes with functional studies, include a protease cleavage site between the tag and protein for post-purification processing.

• Concentration determination: Spectrophotometric methods (e.g., NanoDrop) can be used for protein quantification .

For membrane proteins like CrcB homolog, additional considerations include the possible need for detergent during purification to maintain solubility and proper folding. A systematic screening of detergents (e.g., DDM, LDAO, or OG) at different concentrations should be conducted to identify optimal conditions.

How can researchers design effective experimental controls when studying the function of CrcB homolog?

Designing robust experimental controls is essential for reliable functional characterization of the CrcB homolog. A systematic approach should include:

• Negative controls:

  • Gene deletion mutants: Using CRISPR/Cas9-based systems as described for Shewanella, create a crcB knockout strain to establish baseline phenotypes in its absence .

  • Inactive protein variants: Generate recombinant proteins with mutations in predicted functional domains to serve as non-functional controls.

• Positive controls:

  • Complementation studies: Reintroduce the wild-type crcB gene into knockout strains to confirm phenotype restoration, similar to approaches used for motL studies in Shewanella putrefaciens .

  • Homologous proteins: Include well-characterized CrcB homologs from related species as reference points.

• Expression controls:

  • Western blot analysis using appropriate antibodies to verify protein expression levels, as demonstrated for other Shewanella proteins .

  • Fluorescent protein fusions to monitor localization and expression, with appropriate controls for the tags themselves.

• Environmental controls:

  • Systematic variation of growth conditions to identify contexts where CrcB function becomes apparent or essential.

  • Metal ion availability controls, given the association of Shewanella with metal reduction processes .

These methodological controls will help distinguish specific CrcB-related effects from general physiological responses or experimental artifacts.

What comparative genomic approaches can provide insights into CrcB homolog function across Shewanella species?

Comparative genomic approaches offer powerful insights into CrcB homolog function by leveraging evolutionary relationships and functional conservation. For Shewanella species, researchers should consider:

This multifaceted comparative approach can reveal functional constraints on CrcB evolution and guide hypothesis generation for experimental validation.

How might the CrcB homolog from Shewanella halifaxensis contribute to metal reduction capabilities?

Shewanella species are known for their dissimilatory metal-reducing capabilities, which have important implications for bioremediation and sustainable energy production . While the specific role of CrcB homolog in these processes has not been directly established, several research approaches can explore potential connections:

• Deletion mutant phenotyping: Generate crcB deletion mutants using CRISPR/Cas9-based genome editing and assess changes in metal reduction capabilities across different electron acceptors (Fe(III), Mn(IV), U(VI), etc.).

• Membrane organization studies: Investigate whether CrcB influences the organization of outer membrane cytochromes and other proteins involved in extracellular electron transfer, potentially through membrane domain formation or protein-protein interactions.

• Ion transport considerations: Examine whether CrcB homolog functions in ion transport that might indirectly affect redox homeostasis or metal reduction pathways.

• Expression regulation analysis: Study whether crcB expression is regulated in response to metal availability or redox conditions, which would suggest functional relevance to metal reduction processes.

The highly hydrophobic nature of the CrcB homolog and its predicted membrane localization are consistent with potential roles in organizing membrane components involved in electron transfer or in maintaining ion gradients that support energy conservation during anaerobic respiration.

What are the implications of studying CrcB homolog for understanding antibiotic resistance in Shewanella halifaxensis?

Studying the CrcB homolog in Shewanella halifaxensis may provide insights into antibiotic resistance mechanisms, particularly in light of findings related to other Shewanella strains. Shewanella halifaxensis strain 6JANF4-E-4 has been found to contain an integrative conjugative element of the SXT/R391 family that harbors macrolide resistance determinants mef(C) and mph(G) . While the direct involvement of CrcB homolog in antibiotic resistance has not been established, several research directions could explore potential connections:

• Membrane permeability studies: Investigate whether CrcB affects membrane permeability to antibiotics, particularly hydrophobic compounds.

• Genetic context analysis: Examine the genomic neighborhood of crcB for proximity to known resistance determinants or mobile genetic elements.

• Expression correlation: Analyze whether crcB expression changes in response to antibiotic exposure, which might suggest functional relevance.

• Resistance phenotyping: Compare antibiotic susceptibility profiles between wild-type strains and crcB deletion mutants to identify potential protective effects.

These approaches could reveal whether CrcB homolog contributes to intrinsic antibiotic resistance in Shewanella halifaxensis or interacts with established resistance mechanisms like efflux pumps or membrane permeability barriers.