Recombinant Dictyostelium discoideum Putative uncharacterized transmembrane protein DDB_G0285347 (DDB_G0285347)

What is the molecular structure of DDB_G0285347 and why is it classified as "putative uncharacterized"?

DDB_G0285347 is a 196-amino acid transmembrane protein from Dictyostelium discoideum with the UniProt ID Q54NC9. The protein is classified as "putative uncharacterized" because while bioinformatic analysis suggests it contains transmembrane domains, its specific biological function has not been experimentally verified. The complete amino acid sequence is:

MTIKIRSEETCTESKFFYHNQDVTYNYHLDMVDNGINIWTSIHGKNAGLLPFVFQSFQISSEEDAISFYKYVKLIGTGCYVAILISGNLPYHSKRITKAMKLVGGGSKSIETLSDSNPNFCLIGYKGQKIGSARQAIGDADIEEEGGISVWMMTTKNRCLFKNRILINLRNKTPLGTISQLYKKHIKKEMTNNIYL

Structural prediction algorithms suggest the protein contains hydrophobic regions consistent with membrane insertion, but crystallographic or NMR data is currently unavailable. Researchers should approach functional studies with the understanding that computational predictions require experimental validation.

What conserved domains or motifs are present in DDB_G0285347?

Sequence analysis of DDB_G0285347 reveals several potential functional regions that warrant investigation:

• A potential N-terminal signal sequence (amino acids 1-20)

• Hydrophobic regions consistent with transmembrane domains

• Potential phosphorylation sites at serine residues

• A C-terminal region that may be involved in protein-protein interactions

Researchers should perform multiple sequence alignments with homologous proteins from related species to identify truly conserved residues, as these often indicate functional importance. Domain prediction tools such as PFAM, SMART, or InterPro can provide additional insights into potential functional domains, though experimental validation is essential for confirming these predictions .

How is DDB_G0285347 positioned within Dictyostelium discoideum cellular biology?

As a slime mold, Dictyostelium discoideum serves as an important model organism for studying fundamental cellular processes. Transmembrane proteins like DDB_G0285347 often play crucial roles in cell signaling, adhesion, or transport functions. While the specific cellular localization and function of DDB_G0285347 remain uncharacterized, researchers should consider:

• Expression patterns during different developmental stages of D. discoideum

• Subcellular localization studies using fluorescent tags

• Potential involvement in aggregation, chemotaxis, or differentiation pathways

Understanding the protein's context within D. discoideum biology requires integrated approaches combining gene expression analysis, protein localization, and functional studies .

What is the optimal expression system for recombinant DDB_G0285347 production?

E. coli is the documented expression system for recombinant DDB_G0285347 production. For optimal results, researchers should consider:

• Using BL21(DE3) or Rosetta strains to address potential codon bias issues

• Employing a T7 promoter-based expression vector with an N-terminal His-tag

• Optimizing induction conditions (IPTG concentration, temperature, duration)

• Supplementing with membrane protein expression enhancers when necessary

For transmembrane proteins like DDB_G0285347, expression can be challenging. Alternative expression systems worth considering include yeast (P. pastoris), insect cells (Sf9), or cell-free systems specifically designed for membrane proteins. Each system offers different advantages for maintaining proper folding and post-translational modifications .

What purification strategy yields the highest purity and activity for recombinant DDB_G0285347?

A multi-step purification protocol is recommended for His-tagged DDB_G0285347:

• Cell lysis with appropriate detergents (e.g., n-dodecyl-β-D-maltoside or CHAPS) to solubilize membrane proteins

• Initial capture using Ni-NTA affinity chromatography

• Intermediate purification via ion exchange chromatography

• Polishing step with size exclusion chromatography

Quality control should include SDS-PAGE verification of >90% purity and Western blot confirmation of the target protein. For functional studies, detergent screening is essential to identify conditions that maintain protein stability while allowing biochemical activity .

What analytical methods can confirm proper folding and integrity of purified DDB_G0285347?

To verify that purified DDB_G0285347 maintains its native conformation, researchers should employ:

• Circular dichroism (CD) spectroscopy to assess secondary structure content

• Fluorescence spectroscopy to evaluate tertiary structure

• Dynamic light scattering (DLS) to confirm monodispersity

• Limited proteolysis to assess domain organization and stability

• Thermal shift assays to determine protein stability

For transmembrane proteins, additional techniques such as detergent screening assays and lipid nanodiscs reconstitution can help ensure the protein maintains its native structure. Mass spectrometry can confirm the intact mass and identify any post-translational modifications .

How can researchers investigate potential functional roles of DDB_G0285347?

A comprehensive functional characterization strategy for DDB_G0285347 should include:

• Gene knockout or knockdown studies in D. discoideum to observe phenotypic effects

• Overexpression studies to identify gain-of-function phenotypes

• Localization studies using fluorescently tagged versions of the protein

• Comparative analysis with characterized proteins containing similar domains

• Heterologous expression in mammalian cells to assess conserved functions

Researchers should design experiments that test specific hypotheses about the protein's function based on its sequence features, predicted structure, and expression pattern. For transmembrane proteins, investigating potential roles in signaling, transport, or adhesion is particularly relevant .

What interaction studies would help identify binding partners of DDB_G0285347?

To identify proteins or molecules that interact with DDB_G0285347, consider:

• Co-immunoprecipitation experiments with tagged DDB_G0285347

• Proximity labeling approaches (BioID, APEX) for in vivo interaction mapping

• Yeast two-hybrid screening adapted for membrane proteins

• Pull-down assays with purified recombinant protein

• Crosslinking mass spectrometry to capture transient interactions

For transmembrane proteins, special consideration should be given to maintaining the membrane environment during interaction studies. Detergent selection is critical, and reconstitution into lipid nanodiscs or liposomes may better preserve native interactions .

What structural analysis techniques are appropriate for DDB_G0285347?

Structural characterization of transmembrane proteins presents unique challenges. For DDB_G0285347, consider:

Each technique requires specific sample preparation considerations. For example, cryo-EM may require reconstitution into nanodiscs or amphipols, while crystallography typically requires extensive screening of detergents and crystallization conditions .

What are the optimal storage and handling conditions for recombinant DDB_G0285347?

Based on the product information, recombinant DDB_G0285347 requires careful handling:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution, add glycerol to a final concentration of 50% for long-term storage

• Aliquot the protein to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

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

The protein is supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability. For experimental use, the buffer composition may need optimization depending on the specific application .

How can researchers troubleshoot solubility and stability issues with DDB_G0285347?

Transmembrane proteins like DDB_G0285347 often present solubility and stability challenges. Consider these troubleshooting approaches:

• Screen multiple detergents (DDM, LMNG, CHAPS) at various concentrations

• Test different buffer compositions (pH, salt concentration, additives)

• Evaluate the effect of lipids or cholesterol on protein stability

• Consider protein engineering approaches (truncations, fusion tags)

• Test stabilizing additives such as glycerol, trehalose, or specific ligands

For functional studies, consider reconstituting the protein into lipid nanodiscs, liposomes, or amphipols, which may better mimic the native membrane environment and improve stability .

What controls and validation methods ensure experimental reliability when working with DDB_G0285347?

Rigorous controls are essential for experiments involving uncharacterized proteins:

• Include both positive controls (well-characterized proteins) and negative controls (buffer-only, irrelevant proteins)

• Validate antibody specificity through Western blots comparing wild-type and knockout samples

• Perform rescue experiments in knockout/knockdown models to confirm specificity

• Use multiple independent methods to verify key findings

• Include controls for detergent effects in all biochemical assays

For functional studies, complementary approaches (in vitro biochemistry, cell biology, and in vivo studies) provide the strongest evidence. Always validate key findings with independent techniques .

How can evolutionary analysis of DDB_G0285347 inform functional hypotheses?

Evolutionary analysis provides valuable context for functional studies:

• Perform phylogenetic analysis to identify orthologs in related species

• Compare conservation patterns across different domains of the protein

• Identify lineage-specific adaptations versus universally conserved features

• Analyze selection pressure (dN/dS ratios) across different regions

• Map conserved residues onto structural models to identify potential functional sites

For DDB_G0285347, comparison with homologs in other amoebozoa, as well as more distant eukaryotes, may reveal evolutionary patterns suggesting functional constraints. Analysis of residue conservation in predicted transmembrane regions versus cytoplasmic domains can provide insights into functional importance .

What computational tools can predict structural features and potential binding sites of DDB_G0285347?

Modern computational approaches can provide valuable insights into transmembrane protein structure:

• AlphaFold2 or RoseTTAFold for ab initio structure prediction

• TMHMM or TOPCONS for transmembrane topology prediction

• ScanSite or NetPhos for phosphorylation site prediction

• CASTp or SiteMap for binding pocket identification

• Molecular dynamics simulations to explore conformational flexibility

These predictions generate testable hypotheses about structure-function relationships. Researchers should validate computational predictions through mutagenesis studies targeting predicted functional residues or domains .

How can high-throughput approaches accelerate functional characterization of DDB_G0285347?

Advanced high-throughput methods can efficiently explore multiple hypotheses:

• CRISPR-Cas9 screening to identify genetic interactions

• Phosphoproteomics to map signaling networks

• Metabolomics to identify potential transport substrates

• High-content imaging to assess subcellular localization under various conditions

• Protein microarrays to screen for interaction partners

These approaches generate large datasets that require sophisticated bioinformatic analysis but can rapidly narrow down potential functions. Integration of multiple omics datasets often provides the most comprehensive functional insights .