Recombinant Bovine Transmembrane protein 85 (TMEM85)

Basic Research Questions

• What expression systems are most effective for producing Recombinant Bovine TMEM85?

  Multiple expression systems have been validated for producing recombinant Bovine TMEM85, each with distinct advantages:

  Expression System	Advantages	Considerations
  E. coli	High yield, cost-effective, rapid production	Limited post-translational modifications, potential improper folding
  Yeast	Better folding than bacteria, some post-translational modifications	Moderate yield
  Baculovirus	Higher eukaryotic system, better folding	More complex system, moderate yield
  Mammalian cells	Native-like post-translational modifications, proper folding	Lower yield, higher cost, longer production time

  For structural studies or applications where post-translational modifications are less important, E. coli expression is commonly used . For functional studies requiring properly folded protein with authentic modifications, mammalian expression systems are preferred . Commercially available recombinant bovine TMEM85 is produced using varied systems including E. coli, yeast, baculovirus, and mammalian cells .

• How can researchers validate the functional activity of Recombinant Bovine TMEM85?

  Functional validation of recombinant Bovine TMEM85 should focus on its established anti-apoptotic properties and role in ER membrane protein insertion:

  1. Oxidative stress protection assays:

     • Growth and viability assays in cellular systems exposed to hydrogen peroxide

     • Measurement of cell survival rates with and without TMEM85 expression

     • Quantification of reactive oxygen species levels using fluorescent probes

  2. Membrane protein insertion assays:

     • In vitro reconstitution of membrane insertion using purified components

     • Analysis of insertion efficiency for model tail-anchored proteins

     • Complementation studies in TMEM85/EMC4-deficient cell lines

  Experimental evidence from yeast models demonstrates that both human TMEM85 and its yeast ortholog (YGL231c) significantly increase resistance to oxidative stress . Similar approaches can be used to validate bovine TMEM85, with positive results indicating functional protein.

Advanced Research Questions

Technical Application Questions

• What purification strategies yield the highest purity and activity for Recombinant Bovine TMEM85?

  Purification of recombinant Bovine TMEM85 requires specialized approaches to maintain protein structure and function:

  1. Affinity chromatography methods:

     • Nickel or cobalt affinity for His-tagged versions

     • Optimization of imidazole concentration for elution

     • Consideration of detergent compatibility with affinity resins

  2. Secondary purification steps:

     • Size exclusion chromatography to separate monomeric protein from aggregates

     • Ion exchange chromatography for further purification

     • Gel filtration on Sephadex G75 can be particularly effective for removing endogenous ligands, as demonstrated with other bovine recombinant proteins

  3. Critical parameters:

     • Detergent selection to maintain native structure

     • Buffer composition to ensure stability

     • Temperature conditions during purification

  Commercial preparations of recombinant Bovine TMEM85 typically achieve >90% purity using optimized protocols , and are supplied in stabilizing buffers containing glycerol. For functional studies, researchers should consider additional validation steps beyond purity assessment, including activity assays to ensure the purified protein retains its anti-apoptotic properties.

• How can researchers develop effective antibodies against Bovine TMEM85 for research applications?

  Development of specific antibodies against Bovine TMEM85 requires careful consideration of several factors:

  1. Immunogen selection strategies:

     • Recombinant protein immunization (full-length or domains)

     • Synthetic peptides corresponding to specific regions

     • A successful approach for human TMEM85 used the peptide sequence: LVANRGRRFKWAIELSGPGGGSRGRSDRGSGQGDSLYPVGYLDKQVPDTSVQETDRILVEKRCWDIALGPLKQIPM

  2. Topology considerations:

     • Target accessible regions based on predicted topology

     • Balance between hydrophilic epitopes (better immunogenicity) and specificity

     • Use tools like TMHMM and MEMSAT for topology prediction

  3. Validation methods:

     • Western blotting against recombinant protein and cell lysates

     • Immunoprecipitation to confirm native protein recognition

     • Immunofluorescence for localization studies

     • Knockout/knockdown controls to verify specificity

  4. Cross-reactivity assessment:

     • Testing against TMEM85 from multiple species due to sequence conservation

     • Epitope mapping to identify species-specific regions

  For applications requiring high specificity, monoclonal antibody development is preferred, though validated polyclonal antibodies can offer advantages in detecting native protein conformations. Commercial antibodies against TMEM85 have been successfully used for immunocytochemistry and immunofluorescence applications .

• What strategies can overcome expression and solubility challenges for Recombinant Bovine TMEM85?

  As a multi-pass transmembrane protein, TMEM85 presents significant expression and solubility challenges that can be addressed through various strategies:

  1. Genetic engineering approaches:

     • N-terminal modifications: Similar to the L1A/I2S mutations that facilitate proper processing of other bovine recombinant proteins

     • Codon optimization for expression host

     • Signal sequence optimization

     • Fusion partners to enhance solubility

  2. Expression system optimization:

     • Testing multiple hosts (E. coli, yeast, insect cells, mammalian cells)

     • Evaluation of different promoters and induction conditions

     • Low-temperature expression to promote proper folding

     • Co-expression with chaperones or folding modulators

  3. Solubilization and purification strategies:

     • Detergent screening for optimal extraction

     • Lipid nanodisc or amphipol incorporation

     • Systematic buffer optimization

  4. Stability enhancement:

     • Addition of glycerol in storage buffers (as used in commercial preparations)

     • Single aliquot freeze-storage to avoid freeze-thaw cycles

     • Identification of stabilizing ligands or additives

  For membrane proteins like TMEM85, expression in E. coli often requires specialized strains designed for membrane protein production or strains with oxidizing cytoplasm to promote disulfide bond formation, similar to the approach used for other recombinant bovine proteins where Origami B (DE3) cells have been employed .

• How can researchers design experiments to study the alternative splicing of TMEM85?

  Based on evidence that human TMEM85 undergoes alternative splicing to produce multiple transcripts and proteins , researchers investigating bovine TMEM85 splicing can implement the following experimental approaches:

  1. Transcriptome analysis methods:

     • RNA-Seq to identify splice variants in different bovine tissues

     • RT-PCR with primers spanning potential splice junctions

     • 5' and 3' RACE to characterize transcript ends

  2. Splice variant characterization:

     • Cloning and expression of identified variants

     • Functional comparison of different isoforms

     • Localization studies to determine if variants have different subcellular distributions

  3. Splicing regulation studies:

     • Analysis of tissue-specific expression patterns

     • Investigation of splicing factors that regulate TMEM85

     • Effects of cellular stress on alternative splicing patterns

  4. Functional impact assessment:

     • Comparative anti-apoptotic activity of different isoforms

     • Membrane insertion assays for each variant

     • Protein-protein interaction differences between isoforms

  These approaches can help determine whether bovine TMEM85 undergoes alternative splicing similar to its human counterpart, and whether this contributes to functional diversity in different tissues or under different conditions.

• What experimental models are most suitable for studying Bovine TMEM85 function in dairy cattle applications?

  To investigate the physiological roles of TMEM85 in bovine systems, particularly in contexts relevant to dairy cattle, researchers can employ several model systems:

  1. In vitro models:

     • Bovine mammary epithelial cell lines

     • Primary bovine cell cultures from relevant tissues

     • Bovine tissue explant cultures

  2. Advanced cellular models:

     • 3D organoid cultures of bovine tissues

     • Co-culture systems mimicking tissue microenvironments

     • CRISPR/Cas9-modified cell lines with TMEM85 knockout or modifications

  3. Physiological relevance considerations:

     • Oxidative stress challenges relevant to dairy cattle physiology

     • Models incorporating metabolic stress similar to high-production dairy states

     • Systems investigating potential roles in mammary gland function

  The anti-apoptotic function of TMEM85 may have particular relevance in high-metabolic demand states in dairy cattle, such as during peak lactation when metabolic stress and oxidative challenges are elevated. Understanding these functions could provide insights into cellular resilience mechanisms in production animals, though any research applications would need to consider the regulatory context regarding recombinant bovine proteins in dairy cattle .