While the protein itself is expressed in E. coli, broader insights into recombinant protein optimization can be drawn from studies on CHO cell systems. Key factors influencing expression include:
Codon Optimization: Adjusting codon usage to match host tRNA abundance improves translation efficiency .
Promoter Selection: Strong promoters (e.g., CMV) enhance transcription, though stability may require alternative systems like SV40 .
Post-Translational Modifications: Though E. coli lacks mammalian PTMs, His-tagging simplifies purification for downstream assays .
Despite limited functional data, the protein’s transmembrane topology suggests roles in:
Membrane Signaling: Potential involvement in intercellular communication or environmental sensing.
Transport Processes: Possible participation in ion or nutrient transport across cellular membranes.
Structural Studies: Utilization in cryo-EM or X-ray crystallography to elucidate transmembrane domain interactions .
While no specific pathways or interacting partners are documented for DDB_G0287945, transmembrane proteins often engage in:
Signaling Cascades (e.g., G-protein coupled receptor pathways).
Membrane Remodeling (e.g., interactions with dynamin or actin cytoskeleton components) .
The recombinant protein serves as a tool for:
Functional Screens: High-throughput assays to identify binding partners or enzymatic activity.
Structural Biology: Crystallization studies to resolve transmembrane domain architectures.
Comparative Genomics: Phylogenetic analysis to infer evolutionary roles in Dictyostelium development.
Limited Functional Data: No peer-reviewed studies directly address DDB_G0287945’s role, necessitating further experimental validation.
Host Specificity: E. coli-expressed proteins may lack post-translational modifications critical for native function .
Prioritized studies should include:
Functional Knockout Experiments: Assessing phenotypic changes in Dictyostelium lacking DDB_G0287945.
Protein Interaction Mapping: Co-immunoprecipitation or yeast two-hybrid assays to identify binding partners .
Structural Determination: Solving the crystal structure to inform transmembrane domain interactions.
KEGG: ddi:DDB_G0287945
STRING: 44689.DDB0219294
Dictyostelium discoideum is a social amoeba that can exist as both single cells and in multicellular forms, making it ideal for studying the genetic transitions between unicellular and multicellular life. It has become one of the foremost model organisms for studying fundamental cellular processes including chemotaxis, cytokinesis, phagocytosis, vesicle trafficking, cell motility, and signal transduction . Its genome contains many orthologues of genes associated with human diseases, positioning it as a valuable model for understanding how genetic defects impact normal cell behavior . Additionally, Dictyostelium displays remarkable conservation of DNA repair factors that are targeted in cancer therapies, including poly(ADP-ribose) polymerases used in breast and ovarian cancer treatment .
DDB_G0287945 is a putative uncharacterized transmembrane protein consisting of 68 amino acids. Its full amino acid sequence is MDINKNEINLNRQFSRHVPDEWDFFENSPGENNFLENKSQIRGIFFFFFFFFFFILLILDLIIIIGEL . The protein features a transmembrane domain, suggesting potential roles in membrane-associated processes. The recombinant form used in laboratory settings is typically produced as a full-length protein (amino acids 1-68) with an N-terminal His-tag, expressed in E. coli and provided as a lyophilized powder .
For optimal results, store the lyophilized protein at -20°C/-80°C upon receipt. Prior to opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, add glycerol to a final concentration between 5-50% (with 50% being the standard recommendation) and aliquot to avoid repeated freeze-thaw cycles . Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided to maintain protein integrity .
Dictyostelium exhibits exceptional resistance to DNA damaging agents, including one of the highest known resistances to ionizing radiation . This resistance has been hypothesized to have evolved as a counteractive mechanism against damage incurred during phagocytosis of soil bacteria . Given that Dictyostelium contains orthologues of several DNA repair pathway components otherwise limited to vertebrates , investigating whether DDB_G0287945 interacts with known DNA repair proteins could provide insights into novel aspects of the DNA damage response. Research methodologies should include co-immunoprecipitation studies, DNA damage sensitivity assays in DDB_G0287945 knockout strains, and localization studies following exposure to various DNA damaging agents.
As a transmembrane protein, understanding DDB_G0287945's topology and interaction network is crucial. Researchers should employ a multi-faceted approach including:
Computational prediction of transmembrane domains using tools like TMHMM, Phobius, and TOPCONS
Experimental verification using protease protection assays
Fluorescent protein tagging at N- and C-termini to determine orientation
Proximity labeling techniques (BioID or APEX) to identify neighboring proteins
Split-GFP complementation assays for validation of specific interactions
Super-resolution microscopy to determine precise membrane localization
These approaches would collectively provide a comprehensive understanding of the protein's structural arrangement and potential functional partners within the cellular membrane environment.
Dictyostelium occupies a unique evolutionary position at the crossroads between unicellular and multicellular life . Studying uncharacterized transmembrane proteins like DDB_G0287945 could provide insights into the evolution of membrane-associated signaling pathways that facilitated multicellularity. Comparative genomic analyses across the Amoebozoa phylum, combined with functional studies in Dictyostelium, could reveal whether DDB_G0287945 represents a conserved protein family or a lineage-specific adaptation. Of particular interest would be investigating whether homologues exist in other model organisms and whether functional conservation exists across evolutionary distances.
For creating DDB_G0287945 knockout strains, researchers should consider multiple approaches:
Method | Advantages | Limitations | Validation Approach |
---|---|---|---|
Homologous Recombination | High specificity, stable integration | Time-consuming, requires large homology arms | Southern blot, PCR |
CRISPR-Cas9 | Rapid, efficient, multiplexable | Potential off-targets, PAM site requirements | Sequencing, Western blot |
RNA interference | Allows for partial knockdown, useful if knockout is lethal | Incomplete silencing, variable efficiency | qRT-PCR, Western blot |
The genetic tractability of Dictyostelium makes it amenable to these genetic manipulation techniques. After generation, phenotypic characterization should include growth rate measurements, developmental timing analysis, and stress response assays. If DDB_G0287945 knockout affects DNA repair mechanisms, researchers should test sensitivity to various DNA damaging agents as observed with other DNA repair protein deficiencies in Dictyostelium .
When investigating DDB_G0287945 function, consider these expression systems:
Homologous expression in Dictyostelium:
Most physiologically relevant
Use inducible promoters (e.g., tetracycline-controlled) for temporal control
Enables studies during development and differentiation
Heterologous expression in E. coli:
Mammalian cell expression:
For investigating potential conservation of function
Particularly valuable if human homologues are identified
Each system offers distinct advantages depending on whether the research focus is on protein characterization, interactome analysis, or functional studies in the native cellular environment.
For comprehensive biophysical characterization, researchers should employ:
Circular dichroism (CD) spectroscopy to determine secondary structure content
Nuclear magnetic resonance (NMR) for solution structure of soluble domains
Differential scanning calorimetry (DSC) to assess thermal stability
Surface plasmon resonance (SPR) for binding kinetics with potential interactors
Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions
Cryo-electron microscopy for structural determination in membrane environment
When working with the recombinant protein, researchers should be aware that the His-tag may influence biophysical properties . Control experiments with tag-cleaved versions should be performed when possible.
Dictyostelium's unique life cycle transitions between unicellular and multicellular states make developmental phenotype analysis particularly informative . When analyzing DDB_G0287945 mutant phenotypes:
Distinguish between cell-autonomous defects and those affecting cell-cell communication
Document timing of developmental transitions using time-lapse microscopy
Quantify changes in gene expression for key developmental markers using RNA-seq
Assess cell sorting behaviors in chimeric organisms (mixing mutant and wild-type cells)
Analyze phenotypes across environmental conditions (nutrient availability, pH, temperature)
Interpretation should consider that Dictyostelium development is governed by both cell-intrinsic programs and intercellular signaling . Therefore, phenotypic effects may manifest at either cellular or multicellular levels, requiring multi-scale analysis approaches.
When comparing experimental results across different systems:
Expression level variations:
Account for differences in protein expression levels when comparing phenotypes
Utilize quantitative Western blotting to normalize expression
Post-translational modifications:
Different expression systems may yield proteins with variable modifications
Characterize modifications using mass spectrometry
Experimental conditions:
Standardize buffer conditions, temperature, and pH across systems
Document growth conditions precisely to ensure reproducibility
Data integration:
Develop computational frameworks to integrate data from different experimental approaches
Consider Bayesian statistical approaches for combining evidence from diverse sources
These considerations are particularly important when working with uncharacterized proteins like DDB_G0287945, where initial functional hypotheses may come from diverse experimental systems.
Researchers commonly encounter several challenges when working with this protein:
For recombinant protein work specifically, researchers should carefully follow reconstitution protocols, adding the recommended 5-50% glycerol to prevent aggregation during storage .
Establishing causality in functional studies requires:
Complementation analyses:
Rescue phenotypes with wild-type protein expression
Use point mutants to identify critical functional domains
Acute protein inactivation:
Employ degron systems for rapid protein depletion
Use temperature-sensitive alleles if available
Domain-specific perturbations:
Create chimeric proteins with domain swaps
Utilize structure-guided mutagenesis
Temporal controls:
Use inducible expression/depletion systems
Document phenotypic progression with high temporal resolution
Proximity labeling approaches:
Identify direct interactors using BioID or APEX2 fusion proteins
Validate interactions using reciprocal pulldowns
These approaches collectively strengthen causal inferences by distinguishing primary effects from secondary cellular responses.
Dictyostelium's remarkable resistance to DNA damaging agents and conservation of DNA repair pathways provides a compelling context for studying DDB_G0287945. Future research directions include:
Investigating whether DDB_G0287945 expression changes in response to different DNA damaging agents
Determining if DDB_G0287945 localizes to sites of DNA damage using live-cell imaging
Characterizing genetic interactions between DDB_G0287945 and known DNA repair factors
Assessing whether DDB_G0287945 mutations affect genome stability using mutation accumulation assays
Exploring potential roles in the Fanconi Anemia pathway, which is conserved in Dictyostelium
These investigations could reveal novel connections between membrane proteins and genome maintenance mechanisms, potentially identifying new therapeutic targets for cancer treatment.
Given that Dictyostelium contains many orthologues of genes associated with human diseases , characterizing DDB_G0287945 could have broader implications:
If functional homologues exist in humans, findings could inform understanding of related human proteins
Interactions with conserved signaling pathways might reveal novel regulatory mechanisms
Roles in stress response could illuminate cellular adaptation mechanisms relevant to disease states
If involved in DNA repair, findings could inform cancer therapy resistance mechanisms
Understanding in the context of Dictyostelium's phagocytic behavior could inform immune cell biology
Specifically, researchers should investigate whether DDB_G0287945 affects pathways known to be conserved between Dictyostelium and humans, such as the PARP-mediated DNA damage response targeted in cancer therapies .