Recombinant Macaca fascicularis Transmembrane protein 35 (TMEM35)

How does TMEM35 differ from TMEM35B?

While both are transmembrane proteins, TMEM35 (TMEM35A) and TMEM35B represent distinct proteins with different functions. TMEM35A functions primarily in neuroendocrine and memory processes , whereas TMEM35B has been implicated in pathological processes such as glioma progression . Their expression patterns also differ, with TMEM35B protein mainly expressed in the nucleus rather than membrane compartments . When designing experiments, researchers should be careful not to conflate these two proteins despite their similar nomenclature.

What expression systems are recommended for producing recombinant M. fascicularis TMEM35?

• Prokaryotic systems (E. coli): Suitable for structural studies and antibody production but may lack post-translational modifications

• Eukaryotic systems:

  • Insect cells (Sf9, High Five): Better for membrane proteins with complex folding

  • Mammalian cells (HEK293, CHO): Optimal for functional studies requiring native-like post-translational modifications

When using E. coli, optimizing codons for bacterial expression and employing specialized strains for membrane proteins (e.g., C41/C43) may improve yield and quality.

What are the recommended reconstitution and storage protocols for recombinant TMEM35?

For optimal stability and activity of recombinant M. fascicularis TMEM35:

• Reconstitution:

  • Briefly centrifuge the vial prior to opening

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

  • Add glycerol to a final concentration of 5-50% (50% recommended)

• Storage:

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

  • After reconstitution, aliquot to avoid repeated freeze-thaw cycles

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

  • Long-term storage requires -20°C/-80°C in Tris/PBS-based buffer with 6% trehalose (pH 8.0)

What methodological approaches are effective for studying TMEM35 function in neural tissue?

Based on research methodologies used in related systems, several approaches are recommended:

Knockout studies have been particularly informative, revealing TMEM35's role in HPA axis function, anxiety-related behaviors, and hippocampus-dependent memory consolidation through effects on synaptic plasticity and postsynaptic protein composition .

How can genomic resources for M. fascicularis enhance TMEM35 research?

Recent advances in M. fascicularis genomics provide significant research opportunities:

• High-quality genome assembly: A phased hybrid genomic assembly with chromosome-length scaffolds is now available (European Nucleotide Archive Accession: GCA_902985625) . This resource enables:

  • Precise genetic manipulation strategies

  • Identification of regulatory elements controlling TMEM35 expression

  • Comparative genomic analyses across primate species

• Methodological applications:

  • CRISPR/Cas9 target design with reduced off-target effects

  • Identification of splice variants and isoforms

  • Phylogenetic analysis of TMEM35 evolution in primates

The genomic context of TMEM35 can reveal species-specific regulatory mechanisms that may be relevant when translating findings between model systems .

What are the current challenges in correlating TMEM35 function between rodent models and primate systems?

Several methodological and biological considerations challenge cross-species translation:

• Evolutionary divergence: Despite high conservation, subtle structural differences may affect protein-protein interactions and signaling pathways

• Experimental limitations:

  • Genetic manipulation is more established in mice than primates

  • Direct functional assays (e.g., electrophysiology) in primate brain tissue are technically challenging

• Neuroanatomical differences: While basic HPA axis and limbic circuitry are conserved, important species differences exist in connectivity and cytoarchitecture

• Methodological approaches:

  • Comparative protein-protein interaction studies using co-immunoprecipitation

  • Multi-species phosphoproteomics to identify conserved signaling pathways

  • Cross-species cellular models (e.g., iPSC-derived neurons) to compare function in consistent cellular contexts

How can proteomic approaches identify TMEM35-dependent synaptic changes?

Based on findings from TMEM35 knockout models, quantitative proteomics offers powerful insights:

• Recommended methodology:

  • Synaptosomal fractionation to isolate synaptic compartments

  • Stable isotope labeling (SILAC or TMT) for quantitative comparison

  • Phosphoproteomics to identify changes in signaling pathways

• Critical targets:

  • Postsynaptic density proteins (PSD95 was reduced in knockout models)

  • N-methyl-d-aspartate receptors (NMDARs)

  • Proteins involved in long-term potentiation pathways

• Data analysis approach:

  • Pathway analysis (e.g., Ingenuity Pathway Analysis) to identify molecular networks

  • Correlation with electrophysiological phenotypes

  • Integration with transcriptomic data to identify regulatory mechanisms

What is the significance of TMEM35 in stress-related neurobehavioral research?

TMEM35 knockout studies reveal critical involvement in stress biology:

• Phenotypic effects:

  • Elevated basal corticosterone levels despite normal stress-induced responses

  • Increased anxiety-like behavior

  • Impaired hippocampus-dependent fear and spatial memories

• Research applications:

  • Model for studying mechanisms of anxiety disorders

  • Investigation of memory consolidation pathways

  • HPA axis regulation and dysregulation

• Methodological considerations:

  • Behavioral testing batteries should include both anxiety and memory assessments

  • Corticosterone measurements should include both basal and stress-induced timepoints

  • Molecular analyses should examine both transcriptional and post-translational regulation

How does TMEM35 research contribute to understanding neurodegenerative processes?

While direct evidence from provided sources is limited, TMEM35's role in synaptic plasticity suggests research directions for neurodegenerative studies:

• Synaptic dysfunction: TMEM35 knockout leads to loss of long-term potentiation and reduced levels of proteins critical for synaptic plasticity (PSD95, NMDARs) , which are also implicated in neurodegenerative conditions

• Research approaches:

  • Investigation of TMEM35 expression changes in neurodegenerative disease models

  • Assessment of TMEM35-dependent neuroprotective mechanisms

  • Screening of compounds that modulate TMEM35 function as potential therapeutic targets

• Methodological integration:

  • Combine electrophysiology with molecular analyses

  • Longitudinal studies in aging models

  • Cross-species validation of findings

What are the methodological considerations for using TMEM35 as a research tool in oncology studies?

While TMEM35B (not TMEM35/TMEM35A) has been implicated in glioma biology , researchers should consider:

• Differential diagnostic potential:

  • TMEM35B expression distinguishes between normal tissue and gliomas

  • Expression correlates with tumor stage (higher in stage III+IV than I+II)

  • High diagnostic value based on ROC curve analysis (AUC values)

• Functional assessment methods:

  • siRNA or shRNA knockdown to assess proliferation effects

  • Migration and invasion assays (e.g., Transwell)

  • Analysis of downstream molecular pathways

• Experimental controls:

  • Clear distinction between TMEM35 (TMEM35A) and TMEM35B

  • Appropriate normal tissue controls

  • Validation across multiple cell lines or primary samples

What are common issues in recombinant TMEM35 expression and purification?

When working with recombinant M. fascicularis TMEM35:

• Expression challenges:

  • As a transmembrane protein, TMEM35 may form inclusion bodies in E. coli

  • Protein folding may require specialized conditions

  • Low yield due to toxicity to host cells

• Purification considerations:

  • His-tag accessibility may be limited by protein folding

  • Detergent selection critical for maintaining native conformation

  • Buffer optimization to maintain stability

• Quality control methods:

  • SDS-PAGE to confirm >90% purity

  • Western blotting with anti-His and anti-TMEM35 antibodies

  • Mass spectrometry to confirm protein identity

  • Functional assays appropriate to research questions

How can researchers validate antibodies for TMEM35 detection in M. fascicularis tissues?

Antibody validation is critical for reliable TMEM35 detection:

• Validation methodology:

  • Western blotting against recombinant protein and tissue lysates

  • Immunohistochemistry with peptide competition controls

  • Comparison of multiple antibodies targeting different epitopes

  • Knockout/knockdown controls where available

• Cross-reactivity considerations:

  • Sequence alignment between human, macaque, and mouse TMEM35

  • Epitope conservation analysis

  • Testing in multiple species when possible

• Application-specific validation:

  • For immunohistochemistry: fixation protocol optimization

  • For immunoprecipitation: binding capacity assessment

  • For flow cytometry: cell permeabilization optimization

What analytical methods are recommended for measuring TMEM35 expression levels in experimental systems?

For quantitative analysis of TMEM35 expression:

• mRNA quantification:

  • RT-qPCR with validated primers specific to M. fascicularis TMEM35

  • RNA-Seq for transcriptome-wide context

  • In situ hybridization for spatial distribution

• Protein quantification:

  • Western blotting with appropriate loading controls

  • Immunohistochemistry with standardized scoring systems

  • ELISA development for high-throughput analysis

• Data analysis approaches:

  • Normalization to housekeeping genes/proteins

  • Statistical methods appropriate for data distribution

  • Consideration of biological vs. technical replicates