Recombinant Debaryomyces hansenii UPF0495 protein DEHA2C16280g (DEHA2C16280g)

What are optimal storage conditions for Recombinant DEHA2C16280g protein?

For optimal stability, store the recombinant protein at -20°C/-80°C upon receipt. The protein is typically provided in a Tris/PBS-based buffer with 6% trehalose at pH 8.0. When working with the protein:

• Briefly centrifuge the vial before opening to bring contents to the bottom

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

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

• Aliquot to avoid repeated freeze-thaw cycles

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

How does Debaryomyces hansenii compare to other model yeasts?

Debaryomyces hansenii differs from conventional yeast models like Saccharomyces cerevisiae in several key aspects:

These differences make D. hansenii both challenging to work with and uniquely valuable for specific research applications .

What expression systems are recommended for producing Recombinant DEHA2C16280g?

For recombinant production of DEHA2C16280g, E. coli has been successfully used as an expression host. The following methodological approach is recommended:

• Clone the full-length coding sequence (1-88aa) into an expression vector with an N-terminal His-tag

• Transform into an appropriate E. coli strain optimized for protein expression

• Induce protein expression under standardized conditions

• Purify using immobilized metal affinity chromatography (IMAC)

• Verify protein integrity via SDS-PAGE (expected purity >90%)

Alternative expression systems to consider include:

• Homologous expression in D. hansenii itself using the recently developed CRISPR-Cas9 toolbox

• Heterologous expression in Pichia pastoris for potential glycosylation studies

• Cell-free protein synthesis systems for rapid preliminary studies

How can I design experiments to investigate DEHA2C16280g function?

Since DEHA2C16280g belongs to the UPF0495 family of proteins with unknown function, a systematic experimental approach is recommended:

• Sequence-based analysis:

  • Conduct multiple sequence alignments with homologs from related species

  • Perform domain prediction and structural modeling

  • Identify conserved motifs that might suggest function

• Gene knockout/knockdown studies:

  • Generate a DEHA2C16280g deletion strain using homologous recombination or CRISPR-Cas9

  • Assess phenotypic changes under various growth conditions

  • Compare growth rates, stress responses, and metabolic profiles to wild-type

• Localization studies:

  • Create GFP or other fluorescent protein fusions

  • Determine subcellular localization using high-resolution microscopy

  • Correlate localization with potential functional roles

• Interactome analysis:

  • Perform pull-down assays using the His-tagged recombinant protein

  • Identify interaction partners through mass spectrometry

  • Validate key interactions through co-immunoprecipitation or yeast two-hybrid assays

What transformation methods work best for genetic studies in D. hansenii?

When working with D. hansenii for genetic studies of DEHA2C16280g, consider these optimized transformation protocols:

• Electroporation method:

  • Using a histidine auxotrophic recipient strain (e.g., DBH9) and the DhHIS4 gene as a selectable marker

  • With sorbitol as a stabilizer, transformation efficiency can exceed 1.5 × 10⁵ transformants/μg of DNA

  • This method outperforms earlier protocols that achieved only ~2,000 transformants/μg

• Key components for successful transformation:

  • Utilize autonomous replication sequences (ARS) isolated from D. hansenii

  • DhARS2, DhARS3, and DhARS9 have demonstrated high transformation efficiency

  • CfARS16 from Candida famata (D. hansenii anamorph) is also effective

  • For heterologous gene expression, the TEF1 promoter from Arxula adeninivorans shows strong activity

• In vivo DNA assembly approach:

  • Co-transform up to three DNA fragments with 30-bp homologous overlapping overhangs

  • The fragments will fuse in the correct order in a single step

  • This technique streamlines the generation of transformant strains for high-throughput screening

How might DEHA2C16280g contribute to D. hansenii's osmotolerance?

While the specific role of DEHA2C16280g in osmotolerance has not been directly established, several experimental approaches can be used to investigate this question:

• Physiological characterization:

  • Compare wild-type and DEHA2C16280g knockout strains under varying salt concentrations

  • Measure intracellular ion concentrations, glycerol accumulation, and membrane properties

  • Assess growth rates and viability in high osmolarity conditions

• Transcriptional analysis:

  • Perform RNA-seq to identify genes co-regulated with DEHA2C16280g under salt stress

  • Determine if expression is controlled by known osmostress response pathways

  • Investigate potential regulatory interactions with stress-responsive transcription factors like DhRpn4

• Structural investigations:

  • The protein contains hydrophobic regions (YPLFAAMGVAVASGCFFTY) that may suggest membrane association

  • The charged C-terminal region (EAGNKDEKKEENKD) could be involved in ion interactions

  • Consider how these features might contribute to cellular responses to osmotic stress

What potential relationships exist between DEHA2C16280g and proteasomal regulation?

Given the importance of protein quality control in stress responses and the role of DhRpn4 in proteasomal gene regulation in D. hansenii, potential connections with DEHA2C16280g are worth investigating:

• Transcriptional co-regulation:

  • Determine if DEHA2C16280g expression changes when DhRpn4 is overexpressed or deleted

  • Analyze the DEHA2C16280g promoter region for potential DhRpn4 binding sites

  • Perform ChIP-seq with DhRpn4 to detect potential regulatory interactions

• Protein degradation dynamics:

  • Assess if DEHA2C16280g stability is proteasome-dependent

  • Investigate whether DEHA2C16280g influences the degradation of other proteins

  • Examine potential roles in protein quality control during salt stress

• Stress response coordination:

  • Since DhRpn4 provides resistance to various stresses, examine if DEHA2C16280g functions in similar stress pathways

  • Compare phenotypes of DhRpn4 and DEHA2C16280g mutants under various stress conditions

  • Investigate potential physical interactions between these proteins or their regulatory networks

What challenges should researchers anticipate when working with DEHA2C16280g?

Researchers should be prepared for several technical challenges when studying DEHA2C16280g:

• Protein stability issues:

  • Small proteins (88 amino acids) can be difficult to work with due to stability concerns

  • The hydrophobic regions may cause aggregation during purification

  • Consider fusion tags beyond His-tag (e.g., MBP, GST) to improve solubility

• Functional characterization difficulties:

  • The UPF0495 family has no known function, limiting hypothesis-driven approaches

  • Phenotypic changes in knockout strains may be subtle or condition-dependent

  • Integration of multiple omics approaches may be necessary to reveal function

• Genetic manipulation considerations:

  • While transformation systems exist for D. hansenii, they remain less efficient than for model yeasts

  • Homologous recombination frequency is lower than in S. cerevisiae

  • The slower growth rate of D. hansenii extends experimental timelines

How can high-resolution imaging advance DEHA2C16280g research?

Recent advances in live cell imaging techniques have opened new possibilities for studying D. hansenii proteins including DEHA2C16280g:

• Fluorescent labeling approaches:

  • Combine live cell fluorescent dyes with high-resolution imaging techniques

  • Define subcellular localization of DEHA2C16280g under various conditions

  • Track dynamic changes in protein localization during stress responses

• Holotomography applications:

  • Label-free holotomography has been optimized for visualizing yeast subcellular structures

  • This technique can define physical parameters and visualize membranes and organelles

  • Could reveal how DEHA2C16280g influences cellular architecture during osmotic stress

• Multi-protein tracking:

  • Simultaneous visualization of DEHA2C16280g with other cellular components

  • Correlation of protein localization with organelle inheritance or membrane trafficking pathways

  • Integration with proteomics data to validate interaction networks

What biotechnological applications might emerge from DEHA2C16280g research?

Understanding DEHA2C16280g function could contribute to several biotechnological applications:

• Enhanced halotolerance in industrial strains:

  • If DEHA2C16280g contributes to salt tolerance, its overexpression might enhance industrial strains

  • Engineering improved D. hansenii strains for food fermentation processes

  • Development of salt-tolerant biocatalysts for industrial reactions in high-salt environments

• Biocontrol applications:

  • D. hansenii's halotolerance can inhibit competing microorganisms in industrial processes

  • Understanding how DEHA2C16280g contributes to this property could lead to better biocontrol strategies

  • Applications in food preservation through competitive inhibition of spoilage microorganisms

• Recombinant protein production optimization:

  • Insights into DEHA2C16280g function could improve recombinant protein expression in D. hansenii

  • Development of specialized expression systems for salt-requiring proteins

  • Utilization of industrial side-streams and complex feedstocks for biotechnological applications