Recombinant DDB_G0284989 is a full-length transmembrane protein derived from Dictyostelium discoideum, a slime mold model organism. This protein (UniProt ID: Q54NT8) is annotated as "uncharacterized" due to limited functional data, though its structure and expression parameters are well-documented. It spans 71 amino acids (aa) and contains an N-terminal His-tag for purification and immobilization. The recombinant form is produced in E. coli and retains native-like post-translational modifications absent in prokaryotic systems .
Protein Interaction Studies: His-tag enables immobilization for affinity chromatography or ELISA .
Structural Biology: Serves as a candidate for cryo-EM or NMR studies to resolve transmembrane domains.
KEGG: ddi:DDB_G0284989
DDB_G0284989 is an uncharacterized transmembrane protein from the social amoeba Dictyostelium discoideum, a eukaryotic organism widely used as a model system in biomedical research. This protein, with UniProt ID Q54NT8, is classified as a transmembrane protein, suggesting it spans the cell membrane . Dictyostelium discoideum has been employed as a research model for nearly a century due to its unique life cycle that includes both unicellular and multicellular phases, making it valuable for studying various cellular and developmental processes .
The full-length DDB_G0284989 protein consists of 71 amino acids with the following sequence:
MYKDYLFKSNKGYLSLTLVTLPVCSSLHCYFLWTTLSRLSSLPIDVPRSVCSVASLDLDLVIINLLSILRD
While the detailed three-dimensional structure has not been fully characterized, sequence analysis indicates it is a transmembrane protein. The protein's small size (71 amino acids) suggests it may have a relatively simple structure with potentially a single transmembrane domain. Researchers working with this protein should consider protein structure prediction tools and possibly experimental approaches such as circular dichroism (CD) spectroscopy or nuclear magnetic resonance (NMR) to elucidate its structural features.
In protein databases, DDB_G0284989 is classified as an uncharacterized transmembrane protein from Dictyostelium discoideum with the UniProt ID Q54NT8 . The "uncharacterized" designation indicates that the protein's specific function has not been experimentally determined. The protein is also identified by its DDB_G0284989 gene ID in the Dictyostelium genome database. As research progresses, this classification may be updated to reflect new knowledge about the protein's function or structural characteristics.
The recombinant DDB_G0284989 protein should be stored at -20°C or -80°C upon receipt, with aliquoting necessary for multiple uses to avoid repeated freeze-thaw cycles that could compromise protein integrity . The protein is typically provided as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) and store the aliquoted protein at -20°C or -80°C . Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided to maintain protein functionality .
For reconstitution of recombinant DDB_G0284989, the vial should first be briefly centrifuged to bring the contents to the bottom. The lyophilized protein should then be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage of the reconstituted protein, glycerol should be added to a final concentration of 5-50% (with 50% being the default recommendation), and the solution should be aliquoted to avoid repeated freeze-thaw cycles . The specific reconstitution protocol may need to be optimized depending on the downstream applications, such as functional assays, structural studies, or antibody production.
Expression System | Advantages | Limitations | Best Suited For |
---|---|---|---|
E. coli (bacterial) | High yield, cost-effective, simple culture conditions | May lack post-translational modifications | Basic structural studies, antibody production |
Yeast (S. cerevisiae, P. pastoris) | Eukaryotic processing, some post-translational modifications | Lower yield than bacterial systems | Better folding of eukaryotic proteins |
Insect cells (baculovirus) | More complex post-translational modifications | More complex culture conditions | Functional studies requiring modifications |
Mammalian cells | Most authentic post-translational modifications | Lower yield, higher cost | Studies requiring native protein conformation |
Cell-free systems | Rapid production, no cell culture needed | Limited post-translational modifications | Initial screening, toxic proteins |
The choice should be based on the research requirements, considering factors such as yield, post-translational modifications, and downstream applications.
The search results indicate that recombinant DDB_G0284989 has been produced with an N-terminal His tag , suggesting that immobilized metal affinity chromatography (IMAC) would be an effective primary purification method. A comprehensive purification strategy for this tagged protein might include:
Immobilized Metal Affinity Chromatography (IMAC):
Using Ni-NTA or Co2+ resin to capture the His-tagged protein
Typical binding buffer: 20-50 mM Tris-HCl pH 8.0, 300-500 mM NaCl, 5-20 mM imidazole
Elution with increasing imidazole concentration (250-500 mM)
Size Exclusion Chromatography (SEC):
For further purification and buffer exchange
Separates based on molecular size, useful for removing aggregates
Ion Exchange Chromatography:
If needed for additional purity
Selection of cation or anion exchange based on protein pI
Depending on the specific experimental requirements, additional purification steps or tag removal might be necessary. The purification protocol should be optimized based on protein stability, yield requirements, and the intended downstream applications.
To verify the purity and integrity of recombinant DDB_G0284989, researchers should employ multiple analytical techniques:
SDS-PAGE Analysis:
Western Blot Analysis:
Using anti-His tag antibodies to confirm identity
Alternatively, specific antibodies against DDB_G0284989 if available
Mass Spectrometry:
For exact mass determination and sequence verification
Techniques like MALDI-TOF or ESI-MS
Size Exclusion Chromatography (SEC):
To assess homogeneity and detect aggregation
Circular Dichroism (CD) Spectroscopy:
To evaluate secondary structure and proper folding
A typical purity assessment table might look like:
Analysis Method | Expected Result | Acceptance Criteria |
---|---|---|
SDS-PAGE | Single band at ~8 kDa plus tag | >90% purity |
Western Blot | Positive signal with anti-His antibody | Single band at expected MW |
SEC-HPLC | Single peak | >95% main peak |
Mass Spec | Mass matching theoretical value | ±0.1% of calculated mass |
These methods collectively provide a comprehensive assessment of protein purity, identity, and structural integrity.
To predict the function of DDB_G0284989, researchers should consider:
Conducting computational analyses using tools like InterPro, Pfam, or SMART to identify conserved domains
Performing homology modeling against structurally characterized proteins
Analyzing the presence of signal peptides, transmembrane regions, or other functional motifs
Investigating expression patterns across different developmental stages of Dictyostelium
To investigate potential pathway involvement, researchers could:
Perform knockout or knockdown studies of DDB_G0284989 and analyze resulting phenotypes
Conduct protein-protein interaction studies to identify binding partners
Use phosphoproteomics to detect changes in signaling pathways when DDB_G0284989 is manipulated
Analyze changes in gene expression patterns following DDB_G0284989 perturbation
Since Dictyostelium has been used to study various fundamental cellular processes including cell movement, chemotaxis, differentiation, and autophagy , examining DDB_G0284989's potential role in these processes would be worthwhile.
To study expression patterns, researchers could:
Perform RNA-seq or qRT-PCR analysis at different stages of the Dictyostelium life cycle:
Vegetative growth phase
Starvation response
Aggregation
Mound formation
Slug formation
Culmination and fruiting body formation
Create reporter constructs (e.g., DDB_G0284989 promoter driving GFP expression) to visualize expression patterns in vivo during development
Use Western blotting with specific antibodies to track protein levels throughout development
Understanding expression patterns could provide valuable clues about the protein's function, particularly if it shows stage-specific expression that correlates with specific developmental processes.
To investigate phenotypes associated with DDB_G0284989 disruption, researchers could:
Generate knockout mutants using CRISPR-Cas9 or other gene disruption methods
Analyze mutant phenotypes across various aspects of Dictyostelium biology:
Growth rate in liquid culture and on bacterial lawns
Cell motility and chemotaxis
Response to various chemical compounds or environmental stressors
Developmental progression and morphology
Spore formation and viability
Perform phenotypic rescue experiments by reintroducing the wild-type gene
This systematic phenotypic characterization could provide insights into the protein's biological role and identify potential functional redundancy with other genes.
CRISPR-based gene disruption techniques have been successfully applied in Dictyostelium as mentioned in the search results , providing a powerful tool for studying gene function, including DDB_G0284989. While specific details about CRISPR application for DDB_G0284989 are not provided, a general methodological approach would include:
Guide RNA (gRNA) Design:
Design gRNAs targeting exonic regions of DDB_G0284989, preferably early in the coding sequence
Use Dictyostelium-specific CRISPR design tools to optimize gRNA efficiency and minimize off-target effects
Consider designing multiple gRNAs to increase success rates
CRISPR-Cas9 Delivery:
Construct a vector expressing Cas9 and the selected gRNA(s)
Include appropriate selection markers for Dictyostelium (e.g., G418 resistance)
Transform Dictyostelium cells using electroporation or other established methods
Mutant Selection and Verification:
Select transformants using appropriate antibiotics
Verify gene disruption using PCR, sequencing, and Western blotting
Isolate clonal populations through serial dilution or cloning rings
Phenotypic Analysis:
Compare growth, development, and behavior of mutants to wild-type cells
Conduct specific assays based on hypothesized function of DDB_G0284989
Create rescue strains by reintroducing the wild-type gene to confirm phenotype specificity
This approach would provide valuable insights into DDB_G0284989 function and its role in Dictyostelium biology.
Sequence-Based Homology Searches:
Use BLAST (Basic Local Alignment Search Tool) against comprehensive databases like UniProt, NCBI nr, and organism-specific databases
Employ position-specific iterative BLAST (PSI-BLAST) for detecting remote homologs
Use profile hidden Markov models (HMMs) with tools like HMMER for increased sensitivity
Structural Prediction and Comparison:
Generate structural models using tools like AlphaFold2 or I-TASSER
Use structural alignment tools like DALI or TM-align to identify structural homologs
Analyze conserved structural features that might indicate functional similarity
Phylogenetic Analysis:
Align sequences of potential homologs using tools like MUSCLE or MAFFT
Construct phylogenetic trees to visualize evolutionary relationships
Analyze patterns of sequence conservation across different taxonomic groups
The search results mention that another Dictyostelium protein, GrlJ, shares homology with an uncharacterized human protein, Q8NHA5 , suggesting that similar approaches could identify potential homologs of DDB_G0284989.
Given that the search results identify GrlJ as a G-protein coupled receptor in Dictyostelium that regulates a phenylthiourea-dependent effect , it raises the possibility that other transmembrane proteins like DDB_G0284989 might have similar functions in detecting environmental compounds. To investigate whether DDB_G0284989 might be involved in bitter tastant detection, researchers could:
Comparative Response Assays:
Expose wild-type, grlJ-, and DDB_G0284989- mutant cells to various bitter compounds (phenylthiourea, denatonium benzoate, quinine hydrochloride)
Analyze concentration-dependent cellular responses using motility assays as described for GrlJ
Construct a response profile table comparing the different cell lines:
Compound | Concentration | Wild-type Response | grlJ- Response | DDB_G0284989- Response |
---|---|---|---|---|
Phenylthiourea | 2-5 mM | (baseline) | Partial resistance | ? |
Denatonium benzoate | 1-5 mM | (baseline) | ? | ? |
Quinine hydrochloride | 0.5 mM | (baseline) | ? | ? |
Signal Transduction Analysis:
Structure-Function Studies:
Analyze the transmembrane domain structure of DDB_G0284989 for potential ligand-binding sites
Create chimeric proteins with GrlJ to identify functional domains
Use site-directed mutagenesis to identify critical residues for tastant detection
This systematic approach would help determine whether DDB_G0284989 shares functional characteristics with GrlJ in bitter tastant detection.
Dictyostelium is an established model for studying cell motility and chemotaxis , and the search results show that other transmembrane proteins like GrlJ affect cell movement in response to compounds like phenylthiourea . To investigate potential roles of DDB_G0284989 in cell motility or chemotaxis, researchers could:
Baseline Motility Analysis:
Compare random cell movement parameters between wild-type and DDB_G0284989- cells using time-lapse microscopy
Track key motility metrics:
Cell velocity
Cell directionality
Cell shape changes (aspect ratio)
Pseudopod formation
Actin dynamics
Chemotactic Response Assays:
Test directed migration toward various chemoattractants:
cAMP (primary Dictyostelium chemoattractant)
Folate (bacterial chemoattractant)
Other potential chemoattractants
Gradient Sensing Mechanisms:
Developmental Chemotaxis:
Assess the role of DDB_G0284989 during the aggregation phase of development
Analyze stream formation and mound morphology
Evaluate slug migration and phototactic/thermotactic responses
This comprehensive approach would help determine whether and how DDB_G0284989 contributes to the complex processes of cell motility and chemotaxis in Dictyostelium.
Understanding the subcellular localization of DDB_G0284989 would provide important clues about its function. Based on the search results, Dictyostelium researchers have access to various expression constructs that enable studies on protein localization . To determine the subcellular localization of DDB_G0284989, researchers could employ:
Fluorescent Protein Tagging:
Generate fusion constructs with fluorescent proteins (GFP, RFP, etc.)
Create both N- and C-terminal fusions to account for potential interference with localization signals
Express under native or inducible promoters
Visualize using confocal or super-resolution microscopy
Co-localization Studies:
Perform dual-labeling experiments with known organelle markers:
Plasma membrane: FM4-64 or membrane-targeted fluorescent proteins
Endoplasmic reticulum: ER-Tracker or calreticulin-GFP
Golgi apparatus: golvesin-GFP
Endosomes/lysosomes: RFP-Rab7 or lysotracker
Mitochondria: MitoTracker or mitochondrial-targeted GFP
Immunofluorescence:
Develop specific antibodies against DDB_G0284989
Use anti-tag antibodies if working with tagged versions
Optimize fixation and permeabilization protocols for transmembrane proteins
Subcellular Fractionation:
Separate cellular components through differential centrifugation
Analyze protein distribution across fractions using Western blotting
Create a quantitative distribution profile across cellular compartments
A systematic application of these techniques would provide a comprehensive understanding of where DDB_G0284989 functions within the cell, offering important insights into its biological role.