Recombinant Debaryomyces hansenii Assembly factor CBP4 (CBP4)

What is the optimal expression system for producing recombinant D. hansenii CBP4?

The most effective expression system for recombinant D. hansenii CBP4 is heterologous expression in E. coli. The full-length protein (amino acids 1-142) fused with an N-terminal His tag has been successfully expressed in E. coli systems . When designing your expression construct, consider the following methodological approach:

• Clone the full coding sequence (1-142 aa) into an expression vector with an N-terminal His tag

• Transform into an E. coli expression strain (commercial strains like BL21(DE3) are suitable)

• Induce expression with appropriate conditions (typically IPTG induction or similar method depending on the vector system)

• Grow cultures at lower temperatures (16-25°C) to enhance proper folding of the recombinant protein

This approach yields functional protein with greater than 90% purity as determined by SDS-PAGE analysis .

What is the recommended protocol for reconstitution and storage of lyophilized CBP4?

For optimal reconstitution and storage of lyophilized D. hansenii CBP4, follow this methodological approach:

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

• Reconstitute the 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 recommended as standard)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep aliquots at -20°C/-80°C

Important note: Repeated freezing and thawing significantly reduces protein activity and should be avoided. The storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

How can I design gene targeting experiments to study CBP4 function in D. hansenii?

To study CBP4 function through gene targeting in D. hansenii, you can employ PCR-based gene disruption methods using the following approach:

• Design PCR primers with 50 bp flanking sequences homologous to the regions upstream and downstream of the CBP4 gene

• Amplify a selectable marker cassette (hygromycin B resistance or G418 resistance) using these primers

• Transform the PCR product into D. hansenii cells

• Select transformants on appropriate antibiotic-containing media

• Confirm gene disruption by PCR and/or sequencing

This PCR-mediated gene disruption method has been shown to achieve high efficiency (>75%) integration through homologous recombination in D. hansenii . For the selectable marker, consider using:

When designing your markers, ensure all CTG codons are adapted to alternative leucine codons due to the alternative genetic code in D. hansenii .

What phenotypic assays are appropriate for characterizing CBP4 knockout mutants?

After successfully generating CBP4 knockout mutants, these assays will help characterize the phenotypic effects:

• Growth curve analysis: Monitor growth rates in different media conditions (varying carbon sources, pH, temperature)

• Respiratory capacity assessment: Measure oxygen consumption rates using oxygen electrodes or commercial respiration analysis systems

• Mitochondrial membrane potential: Use fluorescent dyes like JC-1 or TMRM with flow cytometry or fluorescence microscopy

• Salt tolerance testing: Compare growth on media with varying concentrations of NaCl and KCl (0.5M to 2M) to assess potential roles in D. hansenii's halotolerance

• Transcriptome analysis: RNA-seq to identify genes with altered expression in the knockout mutant compared to wild type

Since D. hansenii is known for its halotolerance, special attention should be paid to phenotypic differences under various salt stress conditions, as CBP4 may be involved in adaptation mechanisms to high salt environments .

How does the structure-function relationship of CBP4 contribute to D. hansenii's halotolerance?

Understanding the structure-function relationship of CBP4 in D. hansenii's halotolerance requires a multi-faceted approach:

• Protein structure prediction and analysis: Use bioinformatics tools (AlphaFold, I-TASSER) to predict the tertiary structure of CBP4 and identify functional domains. The transmembrane region (suggested by the amino acid sequence GVVGFSIIATGSLLFKY) may be critical for membrane association in high-salt environments .

• Site-directed mutagenesis: Create point mutations in conserved regions to identify essential residues for function. Follow this experimental approach:

  • Design mutagenic primers with desired base substitutions

  • Perform PCR-based site-directed mutagenesis

  • Express mutant proteins in E. coli

  • Purify and test functional activity in vitro

  • Complement CBP4 knockout D. hansenii with mutant variants

• Comparative functional analysis: Express CBP4 orthologs from non-halotolerant yeasts in D. hansenii CBP4 knockouts to assess functional complementation. Similar approaches have been successful with other D. hansenii genes, such as DhRpn4 .

• Transcriptomic and proteomic integration: Correlate CBP4 expression patterns with global transcriptomic and proteomic changes during salt stress. Previous multi-omics studies show that sodium and potassium trigger different responses at both expression and protein activity levels, suggesting CBP4 may be differentially regulated depending on the specific cation stress .

What role does CBP4 play in mitochondrial function during osmotic stress?

To investigate CBP4's role in mitochondrial function during osmotic stress, implement these methodological approaches:

• Subcellular localization studies:

  • Fuse CBP4 with fluorescent reporters (GFP or mCherry) with a focus on proper codon usage for D. hansenii

  • Visualize localization under normal and osmotic stress conditions

  • Co-localize with mitochondrial markers to confirm mitochondrial association

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation experiments using tagged CBP4

  • Use mass spectrometry to identify interacting partners

  • Validate key interactions with yeast two-hybrid or bimolecular fluorescence complementation

• Mitochondrial functional assays under stress conditions:

  • Measure ATP production in wild-type vs. CBP4 knockout strains

  • Assess cytochrome c oxidase activity

  • Quantify reactive oxygen species (ROS) production using fluorescent probes

• Real-time monitoring of mitochondrial dynamics:

  • Use live-cell imaging to track mitochondrial morphology changes

  • Assess mitochondrial fusion/fission events during osmotic stress

The phosphoproteomic analysis reported in D. hansenii under salt stress conditions revealed changes in protein phosphorylation states, suggesting that post-translational modifications may be important for CBP4 regulation during osmotic stress response .

What are common issues when working with recombinant CBP4 and how can they be resolved?

When working with recombinant D. hansenii CBP4, researchers commonly encounter the following issues and solutions:

When troubleshooting protein purification, monitor each step with SDS-PAGE and optimize elution conditions for your specific construct. For storage, maintaining a final concentration of 50% glycerol has been shown to maximize stability for D. hansenii CBP4 .

How can I overcome challenges in D. hansenii genetic manipulation when studying CBP4?

Genetic manipulation of D. hansenii presents several challenges when studying genes like CBP4. Here are methodological solutions to common issues:

• Low transformation efficiency:

  • Optimize electroporation parameters (voltage, time constant)

  • Use exponentially growing cells in early log phase

  • Pretreat cells with thiol compounds like DTT

  • Increase homology arm length to >50 bp for better integration efficiency

• Multiple gene copies or chromosomal duplications:

  • Screen multiple transformants to identify complete knockouts

  • Use quantitative PCR to determine gene copy number

  • Consider CRISPR-Cas9 approaches for multiple targeting

  • Employ safe harbor sites for heterologous gene expression

• Phenotypic verification challenges:

  • Use complementation tests to confirm phenotype is due to CBP4 disruption

  • Create multiple independent knockout lines to confirm consistent phenotypes

  • Include appropriate controls (wild type, known mutants) in all experiments

• Selective marker limitations:

  • Use heterologous markers like hygromycin B resistance (hph) or G418 resistance (kanr)

  • Ensure markers use appropriate promoters for D. hansenii (S. stipitis TEF1 or ACT1)

  • Remember to adapt CTG codons in marker genes to alternative leucine codons

Recent advances in PCR-based gene targeting techniques have achieved integration efficiencies of >75% for D. hansenii, significantly improving genetic manipulation options for studying genes like CBP4 .

How does D. hansenii CBP4 compare to homologs in other yeast species?

Comparative analysis of D. hansenii CBP4 with homologs in other yeasts reveals interesting evolutionary insights:

• Sequence conservation:
  D. hansenii CBP4 shares moderate sequence identity with homologs in Saccharomyces cerevisiae and other yeasts, with conservation primarily in functional domains. Key differences in transmembrane regions may relate to D. hansenii's adaptation to high-salt environments.

• Functional complementation:
  Similar to the observed cross-species functionality of DhRpn4 (which can activate transcription of proteasomal genes in S. cerevisiae using ScRpn4 binding sites), CBP4 likely retains core functional elements across species while incorporating adaptations specific to D. hansenii's ecological niche .

• Expression regulation:
  Transcription factors like Rpn4 can regulate stress response genes across species. DhRpn4 contains a unique portable transactivation domain (amino acids 43-238) that lacks compact structure but can activate transcription . Similar regulatory patterns might apply to CBP4 expression.

• Evolutionary adaptation:
  The halotolerance of D. hansenii likely shaped the evolution of its proteins, including CBP4. Comparative genomic analyses suggest that proteins involved in stress response show accelerated evolution in D. hansenii compared to non-halotolerant yeasts.

What specific adaptations in CBP4 contribute to D. hansenii's unique environmental tolerance?

D. hansenii's CBP4 likely contains specific adaptations that contribute to the organism's remarkable environmental tolerance:

• Amino acid composition analysis:

  • Higher proportion of acidic residues may enhance stability in high-salt environments

  • Strategic placement of hydrophobic residues in transmembrane domains

  • Potentially fewer Cys residues to reduce oxidative damage susceptibility

• Post-translational modifications:
  The integrated phosphoproteomic analysis of D. hansenii under salt stress revealed specific phosphorylation patterns that may regulate protein activity during stress response . Research methodology to investigate this includes:

  • Phosphoproteome analysis of cells under normal vs. stress conditions

  • Site-directed mutagenesis of phosphorylation sites

  • Functional comparison of phospho-mimetic and phospho-dead variants

• Protein-protein interaction differences:
  CBP4 may interact with different partner proteins in D. hansenii compared to homologs in other yeasts. Experimental approaches include:

  • Comparative interactome analysis using affinity purification-mass spectrometry

  • Yeast two-hybrid screening in different host organisms

  • In vitro binding assays with purified proteins from different species

• Structural adaptations:
  Predicted structural features of D. hansenii CBP4, particularly in membrane-spanning regions, may confer enhanced stability under osmotic stress conditions. This can be investigated through:

  • Computational structure prediction and molecular dynamics simulations

  • Circular dichroism spectroscopy under varying salt conditions

  • Hydrogen-deuterium exchange mass spectrometry to probe structural flexibility