Recombinant Salmonella arizonae Cobalt transport protein CbiN (cbiN)

What are the optimal storage conditions for maintaining CbiN protein stability?

For optimal stability of Recombinant Salmonella arizonae CbiN protein, researchers should store the lyophilized powder at -20°C to -80°C upon receipt. For working solutions, the recommended storage protocol includes:

• Aliquoting the reconstituted protein to minimize freeze-thaw cycles

• Adding glycerol to a final concentration of 50% for long-term storage

• Storing working aliquots at 4°C for no longer than one week

• Avoiding repeated freeze-thaw cycles which can significantly compromise protein integrity

The protein is typically stored in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 for lyophilized powder, or in Tris-based buffer with 50% glycerol for liquid formulations . These conditions have been optimized specifically for this protein to maintain its structural and functional characteristics during storage periods.

What is the recommended reconstitution protocol for lyophilized CbiN protein?

The recommended reconstitution protocol for lyophilized Recombinant Salmonella arizonae CbiN protein involves the following steps:

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

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

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Prepare aliquots for long-term storage at -20°C/-80°C

This protocol helps maintain protein stability while minimizing degradation during the reconstitution process. The addition of glycerol serves as a cryoprotectant that prevents protein denaturation during freeze-thaw cycles . Researchers should note that repeated freezing and thawing is not recommended, and working aliquots should be stored at 4°C for no longer than one week to preserve protein integrity.

How can researchers verify the purity and integrity of recombinant CbiN protein?

Researchers can verify the purity and integrity of recombinant Salmonella arizonae CbiN protein through multiple complementary techniques:

• SDS-PAGE analysis: The standard quality control method confirms purity greater than 90%

• MALDI-TOF MS: This technique provides precise molecular weight verification and can detect post-translational modifications

• Circular dichroism: Useful for confirming proper protein folding and secondary structure

• Size exclusion chromatography: Can detect protein aggregation or degradation products

When performing SDS-PAGE analysis, researchers should observe a primary band corresponding to approximately 10-11 kDa, which represents the expected molecular weight of the CbiN protein with the His-tag. Additional analytical techniques such as Western blotting using anti-His antibodies can further confirm protein identity .

How does CbiN function within the Energy-coupling factor (ECF) transporter system?

The CbiN protein functions as a probable substrate-capture component (S-component) within the Energy-coupling factor (ECF) transporter system in Salmonella arizonae. This transport system facilitates the uptake of cobalt ions, which are essential for various metabolic processes including vitamin B12 biosynthesis.

The functional mechanism involves:

• Initial substrate (cobalt) recognition and binding by the CbiN S-component

• Interaction with other ECF complex components to facilitate transport

• Energy-dependent conformational changes that enable translocation of cobalt across the membrane

• Release of the substrate into the cytoplasm

The CbiN protein's role as an S-component is particularly significant because it provides substrate specificity to the transporter complex . The transmembrane segments of CbiN, identifiable in its amino acid sequence by the hydrophobic regions, are crucial for its membrane integration and function within the transport system. Understanding these mechanisms has implications for bacterial metabolism research and potential antimicrobial target identification.

What experimental approaches can be used to study CbiN protein-protein interactions?

Researchers investigating CbiN protein-protein interactions can employ several sophisticated techniques:

• Pull-down assays: Using the His-tagged CbiN as bait to identify interaction partners

  • Can be combined with mass spectrometry for unbiased partner identification

  • Particularly useful for identifying other components of the ECF transporter system

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics

  • Allows determination of association/dissociation rates and binding affinities

  • Can assess interactions with other transporter components or substrates

• Bacterial two-hybrid systems: For in vivo validation of interactions

  • Allows screening of potential interaction partners in a cellular context

  • Can detect weak or transient interactions that might be missed by in vitro methods

• Crosslinking coupled with mass spectrometry: For capturing transient interactions

  • Provides spatial information about interacting regions

  • Can map the topology of protein complexes

These methodologies provide complementary information about the interaction landscape of CbiN and can help elucidate its functional role within larger multiprotein complexes involved in cobalt transport .

How conserved is CbiN across different Salmonella subspecies and what are the implications for function?

CbiN shows notable conservation patterns across Salmonella subspecies, particularly within the arizonae subspecies. Phylogenetic analysis reveals:

• High conservation of the CbiN amino acid sequence within Salmonella enterica subspecies arizonae

• Conservation of key functional domains involved in substrate binding and transport

• Variation primarily in non-critical regions of the protein

Interestingly, phylogenetic analysis of Salmonella enterica subspecies arizonae using whole-genome sequencing has revealed that certain genetic elements show clade-specific patterns, which may extend to functional elements like the CbiN transport system . This conservation despite the polyphyletic nature of some Salmonella serovars underscores the functional importance of CbiN in bacterial metabolism.

What comparative genomic approaches can be used to study CbiN evolution across bacterial species?

Researchers investigating CbiN evolution can employ several comparative genomic approaches:

These approaches provide a comprehensive evolutionary perspective on CbiN that can inform functional studies and potentially identify novel variants with distinct properties or functions . The integration of these methods with structural analysis can further reveal how sequence conservation relates to functional constraints.

What are the major challenges in expressing and purifying functional CbiN protein?

Researchers face several challenges when expressing and purifying functional CbiN protein:

• Membrane protein solubility issues: As a membrane-associated protein, CbiN may have solubility limitations

  • Solution: Optimize detergent types and concentrations for extraction

  • Use fusion tags (beyond His-tag) that enhance solubility

• Maintaining native conformation: Ensuring the recombinant protein retains its functional structure

  • Solution: Express in bacterial systems that provide appropriate membrane insertion machinery

  • Consider native-like lipid environments during purification

• Low expression yields: Common with membrane-associated proteins like CbiN

  • Solution: Optimize codon usage for expression host

  • Screen multiple expression strains and conditions

  • Consider using specialized expression vectors with strong promoters

• Protein aggregation during purification: Can reduce yield of functional protein

  • Solution: Include stabilizing agents such as glycerol and optimize buffer conditions

  • Perform purification at reduced temperatures (4°C)

  • Consider detergent screening to identify optimal solubilization conditions

These challenges can be addressed through systematic optimization of expression and purification protocols . The current protocols using E. coli as an expression host with N-terminal His-tagging have proven successful, but further refinements may increase yields and functional quality for specific research applications.

How can researchers effectively study the transport function of CbiN in experimental systems?

Studying the transport function of CbiN requires specialized experimental approaches:

• Reconstitution in proteoliposomes: Incorporating purified CbiN into artificial membrane systems

  • Enables direct measurement of transport activities

  • Allows control of membrane composition and environment

  • Can be coupled with fluorescent or radioactive tracer assays to measure cobalt transport

• Whole-cell transport assays: Using genetically modified bacterial strains

  • CbiN knockout strains can establish baseline transport activity

  • Complementation with wild-type or mutant CbiN variants can assess functional significance

  • ICP-MS (Inductively Coupled Plasma Mass Spectrometry) can quantify intracellular cobalt levels

• Electrophysiology: For direct measurement of transport-associated currents

  • Patch-clamp techniques on giant bacterial spheroplasts or reconstituted systems

  • Two-electrode voltage clamp for heterologous expression systems

• Fluorescence-based assays: Using cobalt-sensitive fluorescent probes

  • Real-time monitoring of transport activities

  • Can be adapted for high-throughput screening

Each method has advantages and limitations, and researchers often need to employ multiple approaches to comprehensively characterize transport mechanisms . The choice of system should be guided by the specific research question and available resources.

How can CbiN research be integrated with broader studies on bacterial metal homeostasis?

Integrating CbiN research with broader bacterial metal homeostasis studies requires multidisciplinary approaches:

• Systems biology integration: Placing CbiN function in the context of global cobalt and metal regulatory networks

  • Transcriptomic analysis to identify co-regulated genes

  • Metabolomic studies to link cobalt transport to downstream metabolic pathways

  • Protein-protein interaction networks to map functional relationships

• Comparative metal transport analysis: Examining how different metal transport systems interact

  • Cross-talk between cobalt, nickel, and other metal transport systems

  • Competition studies using multiple metals to assess transport specificity

  • Evaluation of metal-dependent regulatory mechanisms

• Host-pathogen interaction studies: Understanding the role of CbiN in infection contexts

  • Metal sequestration by hosts as an immunity mechanism

  • Bacterial strategies to overcome metal limitation during infection

  • Potential for targeting metal transport systems as antimicrobial strategies

• Structural biology integration: Relating CbiN structure to the broader family of transport proteins

  • Comparative structural analysis with other S-components

  • Structure-based design of inhibitors or substrate analogs

  • Molecular dynamics simulations to understand transport mechanisms

These integrative approaches can provide a comprehensive understanding of how CbiN contributes to bacterial metal homeostasis, potentially identifying new therapeutic targets or biotechnological applications .

What are the potential applications of CbiN in biotechnology and medical research?

CbiN research has several potential applications in biotechnology and medical fields:

• Antimicrobial drug development: CbiN as a potential drug target

  • Inhibition of cobalt transport could disrupt essential bacterial processes

  • Structure-based drug design targeting CbiN-specific features

  • Development of cobalt analogs that could block transport

• Biosensor development: Using CbiN-based systems for metal detection

  • Engineered whole-cell biosensors for environmental monitoring

  • Fluorescence-based reporters coupled to CbiN for cobalt detection

  • Potential applications in water quality assessment

• Vaccine development: Building on knowledge of Salmonella delivery systems

  • CbiN as a potential antigen or component of recombinant vaccines

  • Integration with existing Salmonella-based vaccine delivery platforms

  • Development of attenuated strains with modified metal transport capabilities

• Bioremediation applications: Engineered bacteria with enhanced metal uptake

  • Modified CbiN variants with altered metal specificity or transport rates

  • Applications in heavy metal removal from contaminated environments

  • Potential for metal recovery in industrial processes

The recombinant tools already developed for CbiN research, such as the His-tagged protein expression systems, provide a foundation for these applications . The knowledge gained from fundamental research on CbiN structure and function can be translated into practical solutions for biotechnology and medicine.