Recombinant Dictyostelium discoideum Putative uncharacterized transmembrane protein DDB_G0290657 (DDB_G0290657)
Description
Overview
The Recombinant Dictyostelium discoideum Putative Uncharacterized Transmembrane Protein DDB_G0290657 (DDB_G0290657) is a synthetic version of a predicted transmembrane protein expressed in Dictyostelium discoideum, a social amoeba model organism. The protein is annotated as "uncharacterized" due to limited functional studies, but its structural and biochemical properties have been partially defined through bioinformatics and recombinant production.
Sequence and Molecular Properties
-
Amino Acid Sequence:
MNGPRYAHSNYGGNKGGPSNSTSGVFAGDIRCQVSNKSImLISLKITNSPNSNSRGSSSS SSTSKSSSKTSFTQ -
Theoretical Molecular Weight: ~8.4 kDa (estimated from sequence)
Availability and Production
DDB_G0290657 is commercially available as a recombinant protein, primarily expressed in E. coli with a His-tag for purification. Key production details include:
| Parameter | Details |
|---|---|
| Host Organism | E. coli |
| Tag | His-tag |
| Form | Lyophilized or in solution (Tris-based buffer with 50% glycerol) |
| Storage | -20°C to -80°C; avoid repeated freeze-thaw cycles |
| Price | ~$1,413 for 50 µg |
Organismal Background
Dictyostelium discoideum is a model organism for studying phagocytosis, development, and genome stability . While DDB_G0290657 has not been directly implicated in these processes, its classification as a transmembrane protein suggests potential involvement in:
-
Membrane Trafficking: Phagosome maturation or bacterial engulfment .
-
Signaling Pathways: Response to extracellular cues during multicellular development .
-
Pathogen Interaction: Modulation of immune responses, given D. discoideum’s role as a phagocytic predator .
Knowledge Gaps
-
Functional Studies: No peer-reviewed studies directly investigating DDB_G0290657’s role in D. discoideum biology.
-
Interaction Partners: No reported protein interactions or pathway associations .
-
Evolutionary Conservation: Limited homologs identified outside D. discoideum or related species .
Future Research Directions
To elucidate DDB_G0290657’s function, the following approaches are recommended:
-
Gene Knockout/Overexpression: Assess phenotypic changes in D. discoideum mutants.
-
Proteomic Interactions: Co-IP or crosslinking mass spectrometry to identify binding partners.
-
Functional Assays: Test involvement in bacterial killing (e.g., Klebsiella pneumoniae phagocytosis) .
-
Structural Analysis: X-ray crystallography or cryo-EM to resolve transmembrane domains.
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you require a specific format, please indicate your preference in the order notes. We will fulfill your request whenever possible.
Note: All protein shipments are standardly accompanied by blue ice packs. If you need dry ice shipping, please inform us beforehand, as additional fees will apply.
Generally, the shelf life for liquid form is 6 months at -20°C/-80°C. The shelf life for lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: ddi:DDB_G0290657
Q&A
What are the primary structural characteristics of DDB_G0290657, and how can researchers validate its transmembrane topology?
DDB_G0290657 is predicted to contain 1–2 transmembrane domains (TMDs) via tools like TMHMM and TMpred, with an N-terminal cytoplasmic tail and a C-terminal luminal/extracellular domain . To validate:
-
Membrane Fractionation: Perform carbonate extraction to confirm integral membrane association. Successful partitioning into the membrane pellet after high-pH treatment indicates stable integration .
-
Glycosylation Profiling: Treat purified protein with N-glycosidase F and endoglycosidase H. A molecular weight shift confirms N-glycosylation, while partial endo-H resistance suggests trafficking through the Golgi .
-
Topology Mapping: Use protease protection assays on isolated vesicles. Luminal domains remain intact if protected, while cytoplasmic regions degrade .
Table 1: Predicted Structural Features of DDB_G0290657
Which expression systems are optimal for producing recombinant DDB_G0290657, and what yield can be expected?
Dictyostelium discoideum itself serves as a robust host for secreting recombinant proteins, achieving up to 20 mg/L yields for secreted glycoproteins like PsA . For DDB_G0290657:
-
E. coli Systems: Ideal for initial solubility screening but lack glycosylation capabilities. Use vectors with His-tags for affinity purification .
-
Axenic Dictyostelium Strains: Enable post-translational modifications and secretion. Clone the gene into vectors with constitutive promoters (e.g., actin 15) and validate secretion via Western blot .
-
Mammalian Cells: For functional studies requiring mammalian glycosylation, use HEK293T cells with transient transfection .
How does DDB_G0290657 participate in extracellular vesicle (EV) biogenesis, and what methodologies can track its role in detoxification?
DDB_G0290657 associates with EVs during cellular detoxification, as observed in Dictyostelium resistance to Hoechst 33342 . Key approaches:
-
EV Isolation: Ultracentrifugation (100,000 × g) coupled with sucrose density gradients to separate EVs from apoptotic bodies .
-
Fluorescent Tagging: Fuse DDB_G0290657 with GFP/mCherry and track vesicle release via live imaging under stressors (e.g., DNA-binding dyes) .
-
Lipidomic Profiling: Compare EV membranes from wild-type vs. DDB_G0290657-knockout strains using LC-MS to identify dependency on specific phospholipids .
Table 2: Functional Interactions of DDB_G0290657 in Vesicular Trafficking
| Interaction Partner | Assay Type | Biological Role |
|---|---|---|
| GTPases (e.g., Rab7) | Co-IP + mass spectrometry | Vesicle maturation/lysosomal fusion |
| Detoxification enzymes | Yeast two-hybrid screen | Sequestration of xenobiotics |
What transcriptional regulatory patterns are induced by bacterial encounters, and how does DDB_G0290657 contribute to pathogen response mechanisms?
RNA-seq of Dictyostelium exposed to Bacillus subtilis or Mycobacterium marinum reveals pathogen-specific transcriptional signatures . To dissect DDB_G0290657’s role:
-
Knockout Mutants: Compare RNA-seq profiles of WT vs. ΔDDB_G0290657 strains during bacterial co-culture. Focus on phagocytosis-related genes (e.g., vacuolar ATPases) .
-
CRISPR Interference: Knock down DDB_G0290657 and quantify bacterial survival via colony-forming unit (CFU) assays .
-
Foliate Chemotaxis Assays: Test if DDB_G0290657 modulates folate receptor expression during bacterial hunting .
How can structural homologs like TMEM106B inform functional studies of DDB_G0290657 in lysosomal pathologies?
TMEM106B, a lysosomal type II transmembrane protein linked to frontotemporal dementia, shares topological and functional parallels with DDB_G0290657 . Cross-disciplinary strategies:
-
Chimeric Constructs: Replace TMEM106B TMDs with DDB_G0290657 sequences and assess lysosomal localization in HEK293T cells .
-
Lysosomal pH Measurements: Use pH-sensitive dyes (e.g., LysoSensor) to test if DDB_G0290657 overexpression alters acidification, mimicking TMEM106B dysfunction .
-
Proteomic Profiling: Identify shared interactors using affinity purification-mass spectrometry (AP-MS) in both Dictyostelium and human cell models .
How should researchers resolve discrepancies in subcellular localization reports for DDB_G0290657?
Conflicting localization data (e.g., plasma membrane vs. lysosomes) arise from differential glycosylation or stress-induced trafficking. Mitigation strategies:
-
Context-Specific Staining: Perform immunofluorescence under starvation vs. growth conditions to assess stress-induced relocalization .
-
Glycosylation Mutants: Express asparagine-to-serine mutants at putative glycosylation sites (e.g., N1–N5) and compare localization patterns .
-
Fractionation Controls: Include markers for organelles (e.g., LAMP1 for lysosomes, Na+/K+ ATPase for plasma membrane) in Western blots .
