Recombinant Archaeoglobus fulgidus Cobalt transport protein CbiN (cbiN)

What is Archaeoglobus fulgidus and why is it significant for protein research?

Archaeoglobus fulgidus is a hyperthermophilic archaeon that grows optimally at high temperatures (78-89°C) . It belongs to the domain Archaea and has been extensively studied as a model organism for understanding protein function in extreme environments. The organism's proteins often exhibit exceptional thermostability, making them valuable subjects for structural and functional studies. A. fulgidus has been the focus of various genomic analyses, including heat shock response studies that have revealed important insights into gene regulation under stress conditions . Its adaptations to extreme environments make it particularly interesting for studying specialized proteins like metal transporters.

What is the role of CbiN in A. fulgidus metabolism?

CbiN is part of the cobalt transport system in A. fulgidus, specifically functioning as a component of the energy-coupling factor (ECF) transport system for cobalt uptake. Cobalt is an essential micronutrient required for vitamin B12 (cobalamin) biosynthesis, which serves as a cofactor for various metabolic enzymes. In A. fulgidus, the CbiN protein likely functions alongside other components of the cobalt transport system to facilitate selective uptake of cobalt ions from the environment. While not directly mentioned in the search results, based on similar systems in other organisms, CbiN typically forms part of a substrate-specific module that works with the energizing module of the ECF transporter.

What are the recommended approaches for cloning the cbiN gene from A. fulgidus?

Based on successful cloning strategies used with other A. fulgidus genes, researchers should consider the following approach:

• Design gene-specific primers with appropriate restriction sites, similar to methods used for AF1298 where XhoI and KpnI sites were incorporated .

• Perform PCR amplification using a high-fidelity DNA polymerase such as Platinum Taq high-fidelity DNA polymerase with appropriate cycling conditions .

• Include appropriate buffer components in the PCR reaction (e.g., 6 mM MgCl₂, 150 mM NaCl) .

• Purify the PCR product using gel extraction followed by ligation into an appropriate expression vector such as pBAD/HisA .

• Confirm the correct nucleotide sequence through DNA sequencing before proceeding to expression .

How can I optimize primer design for efficient amplification of cbiN?

For optimal amplification of the cbiN gene:

• Design primers with approximately 20-25 nucleotides complementary to the target sequence.

• Include restriction sites on the 5' ends with 3-6 extra nucleotides before the restriction site to ensure efficient cutting, similar to the approach used for the AF1298 gene (5′-CATATGAAGGGATTAGTGCCCCGAG-3′ and 5′-GCGGCCGCCTTTATCATCCAAACAACTTC-3′) .

• Check primers for self-complementarity and dimer formation potential.

• Ensure the melting temperatures of the primers are compatible (within 5°C of each other).

• Consider the GC content of the primers (aiming for 40-60%).

What expression systems are most effective for recombinant A. fulgidus CbiN production?

Based on successful expression of other A. fulgidus proteins:

• E. coli expression systems using BL21(DE3) derivatives are recommended, particularly BL21codonplus(DE3)-RIL strain which provides additional tRNAs for rare codons that may be present in archaeal genes .

• The pET vector system (such as pET29b) has shown success for archaeal proteins, allowing tight control of expression .

• Including affinity tags such as a histidine tag facilitates subsequent purification, as demonstrated with the rAfung-His fusion protein using TALON Superflow resin .

Table 1: Comparison of Expression Systems for A. fulgidus Proteins

What are the optimal induction conditions for recombinant A. fulgidus CbiN expression?

While specific conditions for CbiN are not directly addressed in the search results, optimal induction conditions can be inferred from successful expression of other A. fulgidus proteins:

• For T7-based systems (pET vectors), IPTG induction at concentrations of 0.5-1 mM is typical.

• Consider temperature adjustment during induction; though A. fulgidus is thermophilic, lower induction temperatures (25-30°C) may improve solubility of the recombinant protein in E. coli.

• Induction time should be optimized; 3-4 hours may be sufficient, but overnight induction at lower temperatures could improve yield for difficult proteins.

• For the pBAD system, L-arabinose at concentrations of 0.02-0.2% is typically used for induction.

What is the recommended purification strategy for recombinant A. fulgidus CbiN?

A multi-step purification approach is recommended:

• Affinity chromatography: For His-tagged CbiN, TALON Superflow resin has proven effective for A. fulgidus proteins .

• Heat treatment: Since A. fulgidus proteins are typically thermostable, a heat treatment step (70-80°C) can be used to remove E. coli host proteins.

• Ion-exchange chromatography: For further purification based on charge differences.

• Size-exclusion chromatography: As a final polishing step to obtain highly pure protein.

Buffer composition is crucial; consider using Tricine or Tris buffer systems (pH 7.5-8.0) containing 150-200 mM KCl or NaCl, and potentially include divalent cations like CaCl₂ (7-20 mM) as used for HSR1 .

How can I assess the purity and integrity of purified recombinant CbiN?

Several analytical techniques should be employed:

• SDS-PAGE (12%) for assessing purity and apparent molecular weight, using appropriate protein standards .

• Western blotting with anti-His antibodies (if His-tagged) following transfer to nitrocellulose membranes .

• Mass spectrometry to confirm the protein identity and integrity.

• Circular dichroism (CD) spectroscopy to evaluate secondary structure integrity.

• Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to assess oligomeric state.

What computational methods can predict CbiN structure in the absence of crystallographic data?

Modern computational approaches include:

• AlphaFold models can be constructed using the ColabFold implementation, as demonstrated with AfAgo protein .

• Structure-based homology detection using HHsearch to identify distant homologs .

• Identification of structurally related proteins using Dali and FoldSeek searches against the PDB .

• Transmembrane region prediction using tools like DeepTMHMM if CbiN contains membrane-spanning segments .

• Structure analysis and visualization with molecular graphics programs like ChimeraX .

How do I identify the cobalt-binding residues in the CbiN protein?

Several complementary approaches can be used:

• Sequence alignment with known cobalt-binding proteins to identify conserved metal-binding motifs.

• Structural analysis to identify potential coordination sites (histidine, aspartate, cysteine residues).

• Site-directed mutagenesis of predicted binding residues followed by functional assays.

• Metal-binding assays such as isothermal titration calorimetry (ITC) or equilibrium dialysis.

• Spectroscopic techniques such as X-ray absorption spectroscopy.

What methods are available for assessing cobalt transport activity of recombinant CbiN?

Functional characterization of CbiN requires:

• Reconstitution into liposomes or proteoliposomes to create a membrane system.

• Radioisotope (⁵⁷Co) uptake assays to directly measure transport activity.

• Fluorescent metal sensors to detect cobalt transport in real-time.

• Membrane potential-sensitive dyes to monitor the coupling of ion gradients to transport.

• In vivo complementation assays using E. coli strains deficient in cobalt transport.

How can protein-protein interactions between CbiN and other components of the cobalt transport system be studied?

Based on techniques used for other A. fulgidus proteins:

• Electrophoretic mobility shift assays (EMSA) could be adapted to study protein-protein interactions, similar to those used for DNA-protein interactions with HSR1 .

• Pull-down assays using His-tagged CbiN to identify interacting partners.

• Bacterial two-hybrid systems to detect interactions in vivo.

• Surface plasmon resonance (SPR) to measure binding kinetics.

• Chemical cross-linking coupled with mass spectrometry to identify interaction interfaces.

How does the hyperthermophilic nature of A. fulgidus affect CbiN structure and function?

The thermophilic nature of A. fulgidus has significant implications for CbiN:

• Proteins from hyperthermophiles typically show enhanced thermostability through increased hydrophobic core packing, additional salt bridges, and decreased surface loop lengths .

• Metal coordination in CbiN may be modified for stability at high temperatures (78-89°C) .

• The protein might exhibit maximum activity at temperatures corresponding to the optimal growth temperature of A. fulgidus.

• Heat shock studies of A. fulgidus have shown that approximately 14% of genes show differential expression under temperature stress , suggesting that metal transport systems may be regulated in response to temperature changes.

How can I study the regulation of cbiN expression in A. fulgidus?

Based on gene regulation studies in A. fulgidus:

• Real-time RT-PCR with gene-specific primers and normalization to a stable reference gene (such as AF0700) .

• Whole-genome microarrays to study expression patterns under different conditions .

• Promoter analysis to identify potential regulatory elements, similar to the approach used for HSR1 binding sites .

• Electrophoretic mobility shift assays (EMSA) to identify potential transcription factors binding to the cbiN promoter .

• DNase I footprinting to precisely map protein-DNA interaction sites .

Why might recombinant A. fulgidus CbiN show low expression levels or poor solubility?

Several factors could contribute to expression challenges:

• Codon bias: Use E. coli strains with additional tRNAs for rare codons, such as BL21codonplus(DE3)-RIL .

• Protein toxicity: Use tightly regulated expression systems and optimize induction conditions.

• Improper folding: Consider co-expression with chaperones or expression at lower temperatures.

• Lack of metal cofactors: Supplement growth media with appropriate metals.

• For integral membrane proteins, consider using specialized membrane protein expression systems.

What approaches can help resolve contradictory results in cobalt binding or transport assays?

When facing contradictory results:

• Verify protein integrity using mass spectrometry and circular dichroism.

• Test multiple binding assay techniques (ITC, fluorescence quenching, equilibrium dialysis).

• Control for buffer components that might interfere with metal binding.

• Compare results under aerobic vs. anaerobic conditions, as cobalt oxidation state may affect binding.

• Perform competition experiments with other divalent metals.

• Consider the oligomeric state of the protein, as it may affect function.