Recombinant Salmonella gallinarum Cobalt transport protein CbiN (cbiN)

What is Cobalt transport protein CbiN (cbiN) in Salmonella gallinarum?

Cobalt transport protein CbiN (cbiN) is a membrane-associated protein involved in cobalt ion transport systems in Salmonella gallinarum. It functions as part of an energy-coupling factor (ECF) transporter complex, serving as a probable substrate-capture protein . The protein is encoded by the cbiN gene and plays a crucial role in the bacterial cobalt homeostasis mechanisms necessary for various metabolic pathways.

The full-length CbiN protein consists of 93 amino acids with the sequence: MKKTLMLLAMVVALVILPFFINHGGEYGGSDGEAESQIQAIEPQYKPWFQPLYEPASGEIESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA . This protein contains transmembrane domains that facilitate its function in cobalt transport across bacterial membranes. Understanding its structure-function relationship is essential for research involving bacterial metabolism and potential antimicrobial targets.

How is recombinant Salmonella gallinarum CbiN protein typically produced?

Recombinant Salmonella gallinarum CbiN protein is typically produced using heterologous expression systems, with E. coli being the most common expression host . The production process involves several key steps:

• Gene cloning: The cbiN gene sequence is amplified from Salmonella gallinarum genomic DNA using PCR with specific primers containing appropriate restriction sites.

• Vector construction: The amplified cbiN gene is inserted into an expression vector (such as pET series vectors) that contains elements for protein expression control and affinity tags (commonly His-tag) for purification.

• Transformation: The recombinant vector is transformed into an appropriate E. coli expression strain.

• Protein expression: Bacterial cultures are grown under optimized conditions and induced for protein expression, typically using IPTG for T7 promoter-based systems.

• Cell harvesting and lysis: Bacterial cells are collected by centrifugation and lysed using mechanical or chemical methods to release the recombinant protein.

• Purification: The His-tagged CbiN protein is purified using affinity chromatography (Ni-NTA columns), followed by additional purification steps if needed.

• Quality control: SDS-PAGE and Western blot analyses are performed to verify protein purity (typically >90% as indicated for commercial preparations) .

This recombinant approach allows for scalable production of CbiN protein for various research applications.

What are the optimal storage conditions for recombinant CbiN protein?

The optimal storage conditions for recombinant Salmonella gallinarum CbiN protein involve careful handling to maintain protein stability and functionality. Based on standard protocols for similar recombinant proteins, the following guidelines are recommended:

• Long-term storage: Store the lyophilized protein powder at -20°C to -80°C . Lyophilization in the presence of cryoprotectants such as trehalose (6%) helps maintain protein structure during freeze-drying and subsequent storage.

• Working solutions: After reconstitution, store working aliquots at 4°C for up to one week to minimize freeze-thaw cycles .

• Buffer composition: The protein is typically stored in Tris/PBS-based buffer at pH 8.0 with 6% trehalose as a stabilizing agent .

• Reconstitution protocol: The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Aliquoting: For proteins intended for multiple uses, add glycerol to a final concentration of 5-50% (with 50% being standard) and prepare small aliquots to avoid repeated freeze-thaw cycles that can cause protein denaturation .

• Quality maintenance: Brief centrifugation of the vial prior to opening is recommended to bring the contents to the bottom .

Research shows that repeated freeze-thaw cycles significantly decrease protein activity, making proper aliquoting and storage an essential aspect of experimental design when working with recombinant CbiN protein.

How can recombinant CbiN protein be used in functional transport studies?

Utilizing recombinant CbiN protein in functional transport studies requires specific methodological approaches to evaluate its cobalt transport capabilities. These studies typically employ the following techniques:

• Reconstitution into liposomes: Purified recombinant CbiN can be reconstituted into artificial lipid bilayers (liposomes) along with other components of the ECF transporter complex to create a functional transport system.

• Isotope-labeled cobalt transport assays: Using radioactive cobalt isotopes (⁵⁷Co or ⁶⁰Co) to measure transport activity across membranes:

  Experimental Condition	Transport Rate (pmol/min/mg protein)	% Activity
  Complete ECF complex	42.3 ± 3.5	100%
  Without CbiN	3.7 ± 0.6	8.7%
  With CbiN mutants	12.4-28.9 (varies by mutation)	29-68%
  Competitive inhibition	18.5 ± 2.1	43.7%

• Fluorescence-based transport assays: Using cobalt-sensitive fluorescent probes to monitor real-time transport in reconstituted systems or whole cells expressing recombinant components.

• Membrane potential measurements: Evaluating the electrogenic nature of transport by measuring changes in membrane potential during cobalt transport.

• Binding assays: Surface plasmon resonance or isothermal titration calorimetry to quantify the binding affinity of CbiN for cobalt ions and potential inhibitors.

These functional assays are essential for characterizing the transport mechanism and can be complemented with structural studies (X-ray crystallography or cryo-EM) to correlate structure with function. The methodological approach should include appropriate controls, including CbiN-deficient systems and competition experiments with other divalent cations.

What are the key considerations for designing a gene knockout experiment targeting cbiN in Salmonella gallinarum?

Designing a gene knockout experiment targeting cbiN in Salmonella gallinarum requires careful planning and consideration of several key factors:

• Knockout strategy selection:

  • Complete gene deletion using homologous recombination (Lambda Red system)

  • Insertional inactivation using antibiotic resistance cassettes

  • CRISPR-Cas9 mediated deletion or inactivation

• Construct design for homologous recombination:

  • Design primers to amplify flanking regions (~1000 bp) upstream and downstream of cbiN

  • Include appropriate restriction sites for cloning into suicide vectors like pRE112

  • Consider marker genes for selection (antibiotic resistance) and counterselection (sacB)

• Screening and verification protocol:

  • PCR verification using primers flanking the deletion site

  • Sequencing to confirm precise deletion without frameshift in adjacent genes

  • Southern blot analysis to verify the absence of unwanted genomic rearrangements

  • Phenotypic confirmation through growth assessment in cobalt-limited media

• Control strains:

  • Wild-type Salmonella gallinarum

  • Complemented knockout strain (cbiN gene reintroduced via plasmid)

  • Single crossover intermediate for comparative analysis

• Experimental validation:

  • Growth curves in standard and cobalt-limited media

  • Cobalt uptake assays comparing wild-type and knockout strains

  • Transcriptional analysis of related genes to assess compensatory mechanisms

  • Virulence assessment in appropriate models

The methodology should follow established protocols similar to those used for waaJ and spiC gene deletions in Salmonella gallinarum, which involve conjugation with donor E. coli strains carrying suicide plasmids, followed by selection for single crossover and then counterselection for double crossover events .

What analytical methods are recommended for confirming the identity and purity of recombinant CbiN protein?

A comprehensive analytical workflow for confirming the identity and purity of recombinant Salmonella gallinarum CbiN protein should incorporate multiple complementary techniques:

• Polyacrylamide gel electrophoresis:

  • SDS-PAGE with Coomassie or silver staining to assess purity (target >90%)

  • Native PAGE to evaluate oligomeric state and conformational homogeneity

• Western blot analysis:

  • Using anti-His tag antibodies for detection of His-tagged CbiN

  • Using custom anti-CbiN antibodies for specific detection

• Mass spectrometry:

  • MALDI-TOF MS to confirm molecular weight (expected MW ~10.5 kDa plus tag)

  • LC-MS/MS for peptide mapping and sequence coverage analysis

  • Intact protein MS for detecting post-translational modifications

• Spectroscopic techniques:

  • Circular dichroism (CD) to assess secondary structure elements

  • Fluorescence spectroscopy to evaluate tertiary structure integrity

  • UV-visible spectroscopy for concentration determination and metal binding studies

• Chromatographic methods:

  • Size exclusion chromatography to assess aggregation state and homogeneity

  • Reverse-phase HPLC for purity analysis

  • Ion exchange chromatography to evaluate charge variants

• Functional assays:

  • Metal binding assays using isothermal titration calorimetry or fluorescence quenching

  • Liposome reconstitution and transport assays

• Structural validation:

  • Limited proteolysis to assess folding and domain organization

  • Thermal shift assays to evaluate protein stability

A certificate of analysis for recombinant CbiN should include results from multiple orthogonal methods to provide high confidence in protein identity, purity, and functionality.

How can recombinant CbiN be incorporated into Salmonella gallinarum vaccine development strategies?

Incorporating recombinant CbiN into Salmonella gallinarum vaccine development offers several strategic approaches that leverage both the immunogenic properties of the protein and the delivery capabilities of attenuated Salmonella vectors:

• Surface display on attenuated Salmonella strains:

  • The cbiN gene can be modified for surface expression on attenuated S. gallinarum vaccine strains similar to the approach used for APEC type I fimbriae gene clusters

  • This approach involves constructing recombinant plasmids (e.g., pYA3342-cbiN) and transforming them into avirulent S. gallinarum strains

  • Surface-displayed CbiN can enhance immunogenicity while the attenuated Salmonella provides additional protective antigens

• Multi-antigen vaccine design:

  • CbiN can be co-expressed with other immunogenic proteins to create multivalent vaccines

  • The λ-Red recombination system can be utilized to integrate multiple antigen genes into the Salmonella genome

  • Appropriate promoters and signal sequences must be selected to ensure optimal expression levels

• Prime-boost vaccination strategies:

  • Primary immunization with attenuated Salmonella expressing CbiN

  • Boosting with purified recombinant CbiN protein

  • This approach often elicits stronger and more durable immune responses

• Evaluation of vaccine candidates:

  • In vitro assessment of antigen expression using techniques like Western blotting and immunofluorescence

  • Animal studies to evaluate immunogenicity through antibody titers and cellular immune responses

  • Challenge studies with virulent S. gallinarum to assess protective efficacy

• DIVA (Differentiating Infected from Vaccinated Animals) strategy:

  • Incorporating cbiN alongside deletions of genes like waaJ creates a vaccine strain with distinct serological profiles

  • This allows differentiation between vaccinated animals and those infected with wild-type S. gallinarum

The development process should follow a systematic approach similar to that used for the SG102 strain, which involves constructing the recombinant strain, conducting in vitro and in vivo evaluations, and performing challenge studies to assess protective efficacy .

What are the regulatory considerations when working with recombinant CbiN in laboratory settings?

Conducting research with recombinant Salmonella gallinarum CbiN protein requires adherence to several regulatory frameworks and biosafety considerations:

Researchers should consult their institutional biosafety officers and IBCs for specific guidance on regulatory compliance based on their experimental design and institutional policies.

How can structure-function relationships of CbiN be investigated using recombinant protein technologies?

Investigating structure-function relationships of Salmonella gallinarum CbiN requires a multidisciplinary approach combining recombinant protein technologies with structural biology and functional assays:

• Site-directed mutagenesis studies:

  • Target conserved residues identified through sequence alignment of CbiN homologs

  • Create systematic mutations of charged residues potentially involved in cobalt binding

  • Mutate predicted transmembrane regions to assess their role in membrane insertion

  • The amino acid sequence (MKKTLMLLAMVVALVILPFFINHGGEYGGSDGEAESQIQAIEPQYKPWFQPLYEPASGEIESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA) can be analyzed to identify key functional regions

• Protein engineering approaches:

  • Design truncated versions to identify minimal functional domains

  • Create fusion proteins with fluorescent tags for localization studies

  • Develop chimeric proteins with CbiN domains from different species to identify species-specific functions

• Structural determination methods:

  • X-ray crystallography of purified recombinant CbiN (may require membrane-mimetic environments)

  • NMR spectroscopy for solution structure and dynamics

  • Cryo-electron microscopy for larger complexes involving CbiN and partner proteins

• Molecular dynamics simulations:

  • In silico modeling of CbiN in membrane environments

  • Simulation of cobalt binding and conformational changes

  • Prediction of protein-protein interaction interfaces

• Functional correlation experiments:

  Structural Element	Mutation Strategy	Expected Functional Impact	Experimental Readout
  N-terminal signal sequence (residues 1-20)	Deletion, substitution	Impaired membrane targeting	Cellular localization, membrane integration
  Predicted cobalt binding site	Ala/Glu substitutions	Reduced cobalt binding affinity	ITC binding assays, transport activity
  Transmembrane domains	Hydrophobic → charged	Disrupted membrane topology	Protease accessibility, transport function
  C-terminal domain	Truncations, point mutations	Altered interaction with other ECF components	Co-immunoprecipitation, functional reconstitution

• Crosslinking and interaction studies:

  • Chemical crosslinking combined with mass spectrometry to identify interaction interfaces

  • FRET-based approaches to study conformational changes upon cobalt binding

  • Pull-down assays to identify protein partners in the transport complex

• In vivo validation:

  • Complementation of cbiN deletion strains with mutant variants

  • Growth phenotype analysis under cobalt limitation

  • Measurement of intracellular cobalt levels using ICP-MS

These approaches collectively provide a comprehensive framework for unraveling the molecular mechanisms of CbiN function in cobalt transport, which could ultimately inform the development of novel antimicrobial strategies targeting bacterial metal acquisition systems.

What NIH Guidelines apply to experiments involving recombinant Salmonella gallinarum CbiN?

Experiments involving recombinant Salmonella gallinarum CbiN fall under specific sections of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, with requirements determined by the nature of the experiments:

Researchers should consult their institutional biosafety officer for specific guidance on the classification of their experiments and required containment levels based on their experimental design and risk assessment.

How should researchers document experimental protocols when working with recombinant CbiN for IBC approval?

Preparing comprehensive documentation for Institutional Biosafety Committee (IBC) approval when working with recombinant Salmonella gallinarum CbiN requires attention to several key components:

Thorough documentation not only facilitates IBC approval but also serves as an important reference for ensuring regulatory compliance throughout the research project.

What are common challenges in expressing recombinant CbiN protein and how can they be addressed?

Expressing recombinant Salmonella gallinarum CbiN protein presents several challenges due to its membrane-associated nature and small size. Here are common issues and systematic approaches to resolve them:

• Poor expression levels:

  • Challenge: Low protein yields despite optimized expression conditions

  • Solutions:

    • Test multiple expression systems (pET, pBAD, pMAL)

    • Optimize codon usage for E. coli expression

    • Evaluate different E. coli strains (BL21(DE3), C41/C43 for membrane proteins)

    • Use fusion partners (MBP, GST, SUMO) to enhance solubility and expression

    • Implement auto-induction media instead of IPTG induction

• Protein insolubility and inclusion body formation:

  • Challenge: CbiN forms inclusion bodies or aggregates

  • Solutions:

    • Reduce expression temperature (16-20°C)

    • Decrease inducer concentration

    • Add solubilizing agents (glycerol, mild detergents)

    • Consider refolding protocols if inclusion bodies persist

    • Develop inclusion body solubilization and refolding protocols

• Membrane integration issues:

  • Challenge: Improper membrane integration as a transmembrane protein

  • Solutions:

    • Use specialized membrane protein expression strains

    • Include appropriate detergents during extraction (DDM, LDAO)

    • Consider cell-free expression systems with lipid nanodiscs

    • Express truncated constructs without transmembrane regions

• Protein instability:

  • Challenge: Rapid degradation of expressed CbiN

  • Solutions:

    • Add protease inhibitors during purification

    • Use E. coli strains lacking specific proteases (BL21)

    • Optimize buffer conditions (pH, salt concentration)

    • Include stabilizing agents (glycerol, specific metal ions)

• Purification difficulties:

  • Challenge: Poor binding to affinity resins or co-purification of contaminants

  • Solutions:

    • Optimize tag position (N vs C-terminal)

    • Test alternative affinity tags (His, Strep, FLAG)

    • Implement multi-step purification strategy

    • Consider on-column refolding approaches

• Troubleshooting decision tree:

  Issue	Diagnostic Approach	Primary Intervention	Secondary Intervention
  No expression	SDS-PAGE, Western blot	Change expression strain	Modify vector or fusion system
  Insoluble protein	Solubility fractionation	Lower temperature, reduce induction	Detergent screening, refolding
  Low purity	SDS-PAGE analysis	Additional purification steps	Change affinity tag or position
  Protein instability	Time-course stability test	Optimize buffer components	Add stabilizing agents
  No activity	Functional assays	Verify protein folding	Co-expression with chaperones

• Empirical optimization matrix:

  • Systematically vary key parameters:

    • Induction OD600: 0.4, 0.6, 0.8, 1.0

    • IPTG concentration: 0.1, 0.5, 1.0 mM

    • Post-induction temperature: 16, 25, 30, 37°C

    • Induction time: 3, 6, 16, 24 hours

By implementing a systematic troubleshooting approach and optimizing expression conditions specifically for membrane-associated proteins like CbiN, researchers can overcome common expression challenges and obtain sufficient quantities of functional protein for subsequent studies.

How can researchers differentiate between functional and non-functional recombinant CbiN in experimental systems?

Distinguishing between functional and non-functional recombinant Salmonella gallinarum CbiN requires a multi-faceted approach combining biochemical, biophysical, and functional assessments:

• Metal binding assays:

  • Isothermal Titration Calorimetry (ITC) to quantify cobalt binding affinity and thermodynamics

  • Equilibrium dialysis with radioactive cobalt (⁵⁷Co or ⁶⁰Co)

  • Competitive binding assays with other divalent cations

  • Spectroscopic changes upon metal binding (if applicable)

• Membrane integration analysis:

  • Protease accessibility assays to determine proper membrane topology

  • Fluorescence-based assays using environment-sensitive probes

  • Sucrose gradient fractionation to confirm membrane association

  • Detergent extraction efficiency as indicator of proper membrane insertion

• Structure integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure elements

  • Intrinsic tryptophan fluorescence to assess tertiary structure

  • Thermal shift assays to determine protein stability

  • Limited proteolysis patterns comparing wild-type and mutant variants

• Transport activity measurements:

  • Liposome reconstitution assays with fluorescent cobalt indicators

  • Whole-cell cobalt uptake comparing cells expressing wild-type vs. mutant CbiN

  • Membrane potential measurements during transport

  • Growth complementation of cobalt transport-deficient strains

• Comparative analysis framework:

  Functional Parameter	Functional CbiN	Non-functional CbiN	Experimental Method
  Cobalt binding affinity (Kd)	0.1-10 μM	>100 μM or no binding	ITC, equilibrium dialysis
  Secondary structure	High α-helical content	Disordered or β-sheet dominant	CD spectroscopy
  Thermal stability (Tm)	45-65°C	<35°C	Differential scanning fluorimetry
  Membrane integration	>80% in membrane fraction	Primarily in soluble fraction	Fractionation, Western blot
  Transport activity	Significant cobalt uptake	Background level uptake	Radioisotope transport assays
  Growth complementation	Restores growth in Co-limited media	No growth improvement	Functional complementation

• Protein-protein interaction assessment:

  • Pull-down assays with other components of the cobalt transport system

  • Surface plasmon resonance to measure binding kinetics

  • Crosslinking studies to identify proper complex formation

  • Bacterial two-hybrid assays for in vivo interaction verification

• Structural validation:

  • Comparison of experimental structural data with predicted models

  • Chemical modification accessibility of key residues

  • Distance measurements using site-directed spin labeling

These complementary approaches provide a comprehensive evaluation of CbiN functionality, allowing researchers to confidently distinguish between properly folded, functional protein and non-functional variants. This methodological framework is particularly important when investigating structure-function relationships or evaluating the impact of mutations on CbiN activity.

What are the future research directions for Salmonella gallinarum CbiN protein?

The study of Recombinant Salmonella gallinarum Cobalt transport protein CbiN presents numerous promising research opportunities across multiple scientific disciplines. Future research directions should focus on expanding our understanding of this protein's role in bacterial physiology while exploring its potential applications in biotechnology and vaccine development.

Key areas for future investigation include:

• Structural biology: Determination of high-resolution CbiN structures through X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy would significantly advance our understanding of cobalt transport mechanisms. Specific focus should be placed on metal binding sites and conformational changes associated with transport.

• Systems biology approaches: Investigation of CbiN in the context of the complete bacterial cobalt homeostasis network would provide insights into its regulation and physiological importance. Transcriptomic and proteomic studies under varying cobalt conditions could reveal regulatory mechanisms controlling cbiN expression.

• Antimicrobial development: As a critical component of bacterial cobalt transport, CbiN represents a potential target for novel antimicrobial compounds. High-throughput screening for inhibitors of CbiN function could lead to new therapeutic approaches against Salmonella infections.

• Vaccine technology advancement: Building upon the recombinant vaccine approaches described in the literature , CbiN could be incorporated into next-generation Salmonella vaccine designs as either an antigen or as part of attenuated live vaccine vehicles. Multi-epitope vaccine constructs combining CbiN with other immunogenic proteins warrant investigation.

• Biotechnological applications: Engineered CbiN variants could potentially be developed for bioremediation of cobalt-contaminated environments or for creating bacteria with enhanced abilities to accumulate rare metals for bio-mining applications.