Recombinant Pongo abelii Transmembrane protein 85 (TMEM85)

What is Pongo abelii Transmembrane protein 85 (TMEM85)?

TMEM85 is a membrane protein found in Sumatran orangutans (Pongo abelii), one of the most endangered great ape species with approximately 6,600 animals remaining in the wild . It belongs to a family of transmembrane proteins characterized by membrane-spanning domains that integrate into cellular membranes. The recombinant form of this protein is produced through molecular cloning techniques, allowing for expression and purification for research purposes. The protein available for research is identified by UniProt accession number Q5RC35 , representing the full-length protein corresponding to amino acids 1-183 of the native sequence.

While the exact function of TMEM85 has not been fully characterized, membrane proteins typically play crucial roles in cellular processes including signal transduction, molecular transport across membranes, cell-cell communication, and structural organization of membrane compartments. Comparative studies with homologous proteins in other species may provide insights into its evolutionary conservation and functional significance.

What are the optimal storage conditions for Recombinant TMEM85?

For maximum stability and preservation of biological activity, Recombinant Pongo abelii TMEM85 should be stored according to the following recommendations:

• Store at -20°C for regular use

• For extended storage, conserve at -20°C or -80°C

• Avoid repeated freezing and thawing cycles, as this can lead to protein denaturation and loss of activity

• For working solutions, store aliquots at 4°C for up to one week maximum

The protein is supplied in a Tris-based buffer containing 50% glycerol, which is optimized for this specific protein's stability . To maximize protein longevity, it is advisable to divide the stock solution into small working aliquots upon receipt, thaw aliquots completely before use, mix gently to ensure homogeneity, and keep on ice when handling at room temperature.

What are the best practices for experimental design when working with TMEM85?

Designing robust experiments with Recombinant TMEM85 requires thoughtful consideration of multiple factors to ensure reliable and interpretable results:

• Control strategies:

  • Include positive controls with known activity/interactions

  • Use negative controls (buffer-only, irrelevant proteins of similar size/type)

  • Consider using a range of protein concentrations to establish dose-dependent relationships

• Validation approaches:

  • Verify protein integrity via SDS-PAGE before experimentation

  • Confirm activity/functionality using established assays where possible

  • Consider western blotting to verify appropriate molecular weight and antibody recognition

• Reproducibility considerations:

  • Perform technical and biological replicates to assess variability

  • Use consistent protein lots when possible for related experiments

  • Document detailed methods including buffer compositions, incubation times, and temperatures

When prior knowledge is limited, researchers should consider implementing robust experimental design (R-ED) principles as described in metabolic flux analysis research . This approach involves systematic exploration of experimental parameters rather than changing multiple variables simultaneously and adopting sampling-based strategies to determine optimal experimental conditions when faced with limited prior information .

How can I optimize protocols for TMEM85 functional studies?

Optimizing protocols for TMEM85 functional studies requires systematic method development and validation:

• Antibody selection and validation:

  • Test multiple antibodies targeting different epitopes of TMEM85

  • Validate antibody specificity using western blot or dot blot analysis

  • Determine optimal antibody concentrations through titration experiments

• Sample preparation optimization:

  • Develop consistent extraction methods appropriate for membrane proteins

  • Evaluate different detergent types and concentrations for solubilization

  • Consider native versus denaturing conditions based on epitope accessibility and experimental goals

• Assay development:

  • Systematically test buffer compositions for optimal protein activity

  • Optimize sample and reagent incubation times and temperatures

  • Determine assay dynamic range and limits of detection

  • Assess intra- and inter-assay variability to establish reproducibility parameters

For interaction studies, it's essential to implement appropriate controls to distinguish specific from non-specific interactions. These should include competition experiments with excess unlabeled protein, testing of mutant variants with altered key residues, and appropriate negative controls using unrelated proteins with similar structural features.

How can robust experimental design principles be applied to TMEM85 research?

Applying robust experimental design (R-ED) principles to TMEM85 research can significantly improve the quality and reliability of results, particularly when prior knowledge is limited. Drawing from methodologies described in metabolic flux analysis research , researchers can implement the following approaches:

• Uncertainty-aware design:

  • Acknowledge limitations in prior knowledge about TMEM85 function

  • Implement a sampling-based approach to explore parameter space rather than relying on a single design based on potentially incorrect assumptions

  • Generate multiple experimental designs and evaluate their expected performance across a range of possible biological scenarios

• Robustification strategies:

  • Design experiments that provide informative results across multiple potential biological realities rather than optimizing for a single hypothesized condition

  • Consider worst-case and average-case performance metrics when evaluating experimental designs

  • Implement bi-level optimization approaches that minimize the maximum expected confidence region of unknown parameters

• Statistical robustness:

  • Utilize the Fisher Information Matrix (FIM) to estimate the expected information content of different experimental designs

  • Apply dimension reduction techniques to focus on the most identifiable parameters

  • Consider condition number thresholds to ensure statistical robustness

By implementing these approaches, researchers can develop experimental designs for TMEM85 studies that are less sensitive to initial assumptions and more likely to yield interpretable results across a range of potential biological scenarios.

How can comparative genomics be applied to TMEM85 research?

Comparative genomics offers powerful frameworks for understanding TMEM85 evolution, function, and species-specific adaptations:

• Evolutionary conservation analysis:

  • Compare TMEM85 sequences across diverse primate lineages and other mammals

  • Calculate evolutionary rates using synonymous/non-synonymous substitution ratios

  • Identify regions under purifying selection (highly conserved) versus positive selection

  • Relate conservation patterns to structural features and putative functional domains

• Population genetics integration:

  • Leverage population structure data from Sumatran orangutans to understand TMEM85 variation

  • Analyze TMEM85 polymorphisms across different orangutan populations separated by geographic barriers such as major rivers, mountain ridges, and the Toba caldera

  • Investigate potential local adaptations in TMEM85 related to different environmental conditions

  • Connect genetic diversity patterns to effective population sizes in different regions

• Synteny and genomic context analysis:

  • Examine the genomic neighborhood of TMEM85 across species

  • Identify conserved gene clusters that might suggest functional relationships

  • Analyze regulatory regions for conserved transcription factor binding sites

  • Compare intron-exon structures to detect alternative splicing conservation

These comparative genomics approaches can provide rich contexts for understanding TMEM85 biology beyond what can be directly observed in experimental systems, particularly in understanding the protein's role in the critically endangered Sumatran orangutan.

What are potential relationships between TMEM85 function and orangutan adaptation?

While specific information about TMEM85's role in orangutan adaptation is not directly provided in the search results, we can outline research directions based on what we know about membrane proteins and Sumatran orangutan ecology:

• Population structure considerations:

  • The marked population structure of Sumatran orangutans, influenced by geographical barriers like rivers and mountain ridges , may have implications for TMEM85 genetic diversity

  • Limited effective population sizes in isolated subpopulations could affect the fixation of TMEM85 variants

  • Male-driven long-distance gene flow patterns might impact the distribution of TMEM85 variants differently than maternally inherited genes

• Functional adaptation hypotheses:

  • Membrane proteins often play roles in cellular defense, pathogen recognition, and environmental adaptation

  • TMEM85 could potentially be involved in adaptations to specific dietary components, pathogens, or environmental challenges faced by orangutans

  • Changes in protein-protein interactions or subcellular localization might reflect evolutionary adaptations

• Conservation implications:

  • Understanding the genetic diversity of functional genes like TMEM85 across orangutan populations provides insights for conservation strategies

  • Genetic variation in such genes may contribute to the adaptive potential of endangered populations

  • Conservation efforts might benefit from maintaining diverse TMEM85 variants in managed populations

Research exploring these dimensions of TMEM85 would require integration of molecular evolution approaches with functional studies and population genetics data from the critically endangered Sumatran orangutan populations.

What methodological approaches can be used to investigate TMEM85 membrane dynamics?

While specific information about TMEM85's role in membrane dynamics is not directly provided in the search results, several methodological approaches can be employed to investigate this aspect:

• Subcellular localization studies:

  • Employ fluorescently tagged TMEM85 constructs to visualize localization in live cells

  • Use immunofluorescence with specific antibodies against native TMEM85

  • Perform subcellular fractionation followed by western blotting to determine membrane compartment distribution

  • Utilize proximity labeling approaches (BioID, APEX) to identify the local proteome environment

• Membrane topology analysis:

  • Determine the orientation of TMEM85 within membranes using protease protection assays

  • Apply glycosylation site mapping to identify luminal domains

  • Use cysteine accessibility methods to probe transmembrane domain organization

  • Develop epitope insertion strategies to map domain exposure

• Dynamic behavior investigation:

  • Implement fluorescence recovery after photobleaching (FRAP) to measure lateral mobility

  • Apply single-particle tracking to analyze diffusion characteristics

  • Use fluorescence resonance energy transfer (FRET) to detect protein-protein interactions

  • Employ optogenetic approaches to manipulate TMEM85 activity in real-time

Through systematic application of these methodologies, researchers can build a comprehensive understanding of TMEM85's contribution to membrane dynamics and related cellular processes.

How should contradictory results in TMEM85 studies be interpreted?

When facing contradictory results in TMEM85 functional assays, a systematic troubleshooting and interpretation approach is essential:

• Methodological reconciliation:

  • Carefully compare experimental protocols for subtle differences that might explain discrepancies

  • Evaluate reagent sources, preparation methods, and quality control procedures

  • Consider differences in detection methods, sensitivity, and dynamic ranges

  • Assess whether contradictions arise from different experimental models or conditions

• Statistical reassessment:

  • Determine if contradictions are statistically significant or within expected variability

  • Evaluate sample sizes and statistical power in each experiment

  • Consider applying more sophisticated statistical methods appropriate for the data structure

  • Implement meta-analysis approaches if multiple datasets are available

• Biological complexity considerations:

  • Explore whether contradictions might reflect genuine biological complexity

  • Consider context-dependent functions in different cell types or physiological states

  • Evaluate potential post-translational modifications or conformational states

  • Assess whether protein interaction partners present in one system but not another could explain differences

When analyzing contradictory results, it's important to maintain scientific rigor while being open to unexpected biology. The approaches outlined in research on robust experimental design provide valuable frameworks for navigating uncertainty and designing experiments that yield informative results even when prior knowledge is limited.

What are common pitfalls in recombinant membrane protein studies relevant to TMEM85?

Working with recombinant membrane proteins like TMEM85 presents unique challenges. Recognizing and addressing these common pitfalls can significantly improve research outcomes:

• Expression system selection issues:

  • Pitfall: Choosing inappropriate expression systems for membrane proteins

  • Solution: Consider specialized systems optimized for membrane proteins such as mammalian expression systems for proper folding and post-translational modifications, insect cell systems for higher yields, or cell-free systems supplemented with lipids

• Protein solubility and extraction problems:

  • Pitfall: Inadequate solubilization leading to aggregation or denaturation

  • Solution: Screen multiple detergents for efficiency and preservation of structure; consider native nanodiscs or styrene maleic acid lipid particles (SMALPs) for detergent-free extraction; optimize buffer conditions (pH, salt, glycerol) to enhance stability

• Protein folding and trafficking challenges:

  • Pitfall: Improper folding or cellular processing in heterologous systems

  • Solution: Include molecular chaperones or folding enhancers; consider fusion partners that promote membrane insertion; optimize growth/induction conditions; verify cellular localization

• Storage and stability issues:

  • Pitfall: Protein degradation or activity loss during storage

  • Solution: Follow recommended storage protocols (-20°C/-80°C in appropriate buffer) ; avoid repeated freeze-thaw cycles by preparing single-use aliquots; include stability enhancers (glycerol, specific lipids) in storage buffers

By anticipating these common challenges and implementing appropriate strategies, researchers can enhance the success rate and reliability of TMEM85 recombinant protein studies.

What statistical approaches are most appropriate for analyzing TMEM85 interaction data?

Selecting appropriate statistical methods for analyzing TMEM85 interaction data depends on the experimental approach and data characteristics:

• For interaction screening data:

  • Implement multiple testing correction (Benjamini-Hochberg, Bonferroni) for high-throughput screens

  • Apply appropriate normalization methods for technology-specific biases

  • Utilize robust Z-score or similar metrics to identify significant hits

  • For targeted studies, apply hypothesis testing based on data distribution and implement power analysis to ensure adequate sample size

• For quantitative interaction analysis:

  • Apply appropriate binding models (one-site, two-site, cooperative) for affinity data

  • Use non-linear regression for KD determination

  • Implement global fitting for complex binding scenarios

  • Calculate confidence intervals for binding parameters

• For network analysis approaches:

  • Apply graph theory metrics (centrality, clustering coefficient) for interactome mapping

  • Implement community detection algorithms to identify functional modules

  • Use permutation-based approaches to assess network significance

  • Apply statistical tests for network overlap significance in comparative studies

• For integration with multi-omics data:

  • Apply canonical correlation analysis for multi-dataset relationships

  • Implement integrative clustering approaches

  • Consider tensor decomposition methods for multi-way data

  • Use Bayesian integration frameworks for complex data integration

When analyzing experimental design strategies, researchers might also consider applying automated experimental design approaches with optimization from historical data simulations, as described in recent research on experimental design optimization .

How might TMEM85 research contribute to orangutan conservation efforts?

TMEM85 research has potential implications for conservation of the critically endangered Sumatran orangutan:

• Genetic diversity assessment:

  • Understanding genetic diversity in functional genes like TMEM85 across orangutan populations provides insights into the adaptive potential of these endangered populations

  • Analysis of TMEM85 variants may help identify genetically distinct subpopulations requiring specialized conservation approaches

  • Such research could contribute to understanding how the pronounced population structure, caused by geographic barriers , affects genetic diversity at functional loci

• Adaptation and resilience insights:

  • Research on TMEM85 function may reveal adaptations specific to the unique environmental challenges faced by different orangutan populations

  • Understanding the molecular basis of adaptation could inform predictions about population resilience to environmental changes

  • Insights into male-driven gene flow patterns and their impact on functional genetic diversity could inform conservation management strategies

• Conservation genomics applications:

  • TMEM85 could serve as one of many markers for monitoring genetic health in wild and captive populations

  • Functional genetic diversity assessment could complement neutral marker studies traditionally used in conservation genetics

  • Understanding how fragmentation affects functional genetic diversity could inform corridor design and reconnection strategies

By linking molecular research on proteins like TMEM85 with conservation biology, researchers can develop more comprehensive approaches to preserving not just the species but also its adaptive potential and evolutionary future.