Recombinant Bovine Transmembrane protein 183 (TMEM183)

What is Bovine Transmembrane Protein 183 and why is it significant in research?

Bovine Transmembrane Protein 183 (TMEM183) is a membrane-spanning protein found in bovine cells that plays potential roles in cellular signaling and membrane transport. Similar to other recombinant bovine proteins, TMEM183 is significant for understanding cellular mechanisms and potential biomarker applications. Research approaches for TMEM183 can be informed by methodologies used for other recombinant bovine proteins, which typically involve expression in bacterial systems, purification using affinity chromatography, and functional characterization through various biochemical and biophysical techniques .

What expression systems are most effective for recombinant bovine TMEM183?

For transmembrane proteins like TMEM183, bacterial expression systems utilizing E. coli strains such as Lemo21 (DE3) are commonly employed. This strain allows for controlled expression through IPTG induction. Based on protocols for other recombinant bovine proteins, optimal expression conditions would include:

• Transformation into E. coli Lemo21 (DE3)

• Culture on LB agar with appropriate antibiotics (typically kanamycin at 50 μg/mL)

• Incubation at 30°C for 12-16 hours

• Inoculation of single colonies into LB broth with antibiotics

• Induction at OD600 of 0.4 with IPTG (0.4 mM final concentration)

• Post-induction incubation for 3 hours at 30°C with 200 rpm shaking

For membrane proteins that show poor expression, lower temperature incubation (16°C for 18 hours) may significantly improve yields, as demonstrated with other recombinant proteins .

How do you confirm successful expression of recombinant bovine TMEM183?

Confirmation of successful TMEM183 expression requires:

• SDS-PAGE analysis to visualize protein bands at the expected molecular weight

• Western blotting using anti-His Tag antibodies (if a His-tag was incorporated)

• Visualization using chemiluminescence imaging systems

• Mass spectrometry confirmation of protein identity

Protein identification through LC-MS/MS is particularly valuable, involving:

• Excision of the protein band from SDS-PAGE

• In-gel trypsin digestion

• Peptide extraction and analysis using LC-MS/MS

• Database searching against UniProt or NCBI using software like Mascot

• Confirmation based on peptide matches with P-values less than 0.05

What strategies address the typical insolubility of bovine transmembrane proteins like TMEM183?

Transmembrane proteins frequently form inclusion bodies during recombinant expression. Based on studies with other recombinant bovine proteins, effective solubilization strategies include:

For TMEM183, PCL lysis buffer containing strong detergents would likely be most effective, as demonstrated with other membrane-associated proteins that remain primarily in the insoluble fraction with milder lysis conditions .

What purification methods yield the highest purity for recombinant bovine TMEM183?

For transmembrane proteins like TMEM183, purification typically involves:

• Anionic denaturation of inclusion bodies

• Affinity chromatography (typically His-tag based)

• Gradient elution with increasing imidazole concentrations

• SDS-PAGE and Western blot confirmation of purified fractions

• Desalting using centrifugal filter units (30 kDa molecular weight cutoff)

• Protein concentration determination using Bradford or similar assays

Researchers should be vigilant about potential degradation after purification, as observed with some recombinant bovine proteins that showed significant degradation within 48 hours of purification .

How can protein stability of purified TMEM183 be assessed and enhanced?

Stability assessment for TMEM183 should include:

• Time-course analysis of purified protein using SDS-PAGE and Western blotting

• Testing storage conditions (temperature, buffer composition, additives)

• Monitoring degradation patterns

Potential stability enhancers include:

• Addition of protease inhibitors

• Storage at -80°C in small aliquots

• Addition of glycerol (10-20%) to storage buffer

• Use of reducing agents like DTT or β-mercaptoethanol

• Buffer optimization for pH and salt concentration

Some recombinant bovine proteins showed significant degradation within 48 hours after purification, suggesting that immediate use or proper stabilization methods are crucial .

What analytical techniques are most informative for characterizing recombinant bovine TMEM183?

Multiple analytical techniques provide complementary information about TMEM183:

• Protein Identification:

  • LC-MS/MS analysis following tryptic digestion

  • Database searching with parameters including:

    • Parent ion mass tolerance: 10 ppm

    • MS/MS ion mass tolerance: 0.02 Da

    • Fixed modifications: Carbamidomethylation of cysteine

    • Variable modifications: Oxidation of methionine, pyro-Glu formation, deamidation of asparagine and glutamine

• Structural Analysis:

  • Circular dichroism for secondary structure evaluation

  • Size-exclusion chromatography for oligomeric state

  • Limited proteolysis for domain identification

• Functional Analysis:

  • Binding assays with potential interaction partners

  • Transport assays if relevant to function

  • Antibody recognition tests

How can recombinant bovine TMEM183 be used as a biomarker in research applications?

While specific information about TMEM183 as a biomarker is not available, approaches can be informed by other recombinant bovine protein biomarker studies:

• Develop specific antibody-based detection methods (ELISA, multiplexed immunoassays)

• Establish baseline expression levels in various bovine tissues

• Investigate concentration changes under different physiological conditions

• Assess stability and half-life in biological samples

• Evaluate specificity and sensitivity as a biomarker

When developing biomarker applications, it's important to consider:

• Inter-individual physiological differences affecting baseline levels

• Factors influencing expression (age, physiological state)

• Responders vs. non-responders in experimental conditions

• Decision limits for biomarker positivity based on reference populations

What considerations are important when designing multiplex assays including TMEM183?

For multiplex assays incorporating TMEM183 alongside other proteins:

• Ensure antibody specificity with minimal cross-reactivity

• Optimize buffer conditions compatible with all target proteins

• Establish individual decision limits for each biomarker

• Consider additive biomarker analysis approaches for improved predictive power

• Account for inter-individual differences in baseline levels

• Calculate true-positive and false-positive rates for assay validation

Research with other bovine protein biomarkers demonstrates that combining multiple markers often provides better predictive power than single markers alone. In some studies, while individual biomarkers failed to reach the targeted 95% true-prediction rate, combining biomarker results significantly improved detection capabilities .

How should experiments be designed to study TMEM183 expression changes under different conditions?

Effective experimental design for TMEM183 expression studies should include:

• Study design elements:

  • Clear adaptation/baseline period (typically 2 weeks)

  • Well-defined treatment periods

  • Appropriate withdrawal periods

  • Inclusion of untreated control animals

  • Sufficient biological replicates (minimum n=6 per group)

  • Time-course sampling to capture expression dynamics

• Controls and reference points:

  • Establishment of individual baseline levels

  • Decision limits based on reference populations

  • Inclusion of positive and negative controls

  • Age-matched animals when possible

• Data analysis approaches:

  • Time-course expression profiles

  • Calculation of true-positive and false-positive rates

  • Statistical methods accounting for inter-individual variations

  • Integration of multiple biomarkers when relevant

What are common pitfalls in recombinant bovine TMEM183 expression and how can they be addressed?

Based on experiences with other recombinant bovine proteins, common challenges include:

• Poor expression levels:

  • Solution: Optimize codon usage for E. coli

  • Solution: Test lower temperature induction (16°C for 18h)

  • Solution: Evaluate alternative expression strains

• Protein insolubility:

  • Solution: Use stronger lysis buffers containing SDS or other strong detergents

  • Solution: Develop refolding protocols from inclusion bodies

  • Solution: Consider membrane-mimetic environments for refolding

• Protein degradation:

  • Solution: Add protease inhibitors during purification

  • Solution: Minimize processing time

  • Solution: Optimize storage conditions immediately after purification

• Non-specific antibody binding:

  • Solution: Validate antibody specificity

  • Solution: Optimize blocking conditions

  • Solution: Use alternative detection methods

How do you interpret contradictory results when analyzing TMEM183 expression data across different studies?

When faced with contradictory results across studies:

• Examine differences in experimental protocols:

  • Expression systems and conditions

  • Purification methods

  • Detection techniques

  • Sample preparation procedures

• Consider biological variables:

  • Age differences among study animals (younger vs. older animals may show different response patterns)

  • Physiological state variations

  • Genetic backgrounds

  • Environmental factors

• Analyze methodological differences:

  • Antibody specificity and sensitivity

  • Decision limit calculations

  • Data normalization approaches

  • Statistical methods employed

Research with other bovine proteins has shown that factors such as age can significantly affect experimental outcomes. For example, in studies of bovine somatotropin biomarkers, antibody responses tended to be higher in older animals, while responses in younger animals declined more quickly .

How can site-directed mutagenesis of TMEM183 help elucidate structure-function relationships?

Site-directed mutagenesis offers powerful insights into TMEM183 function:

• Target selection strategies:

  • Conserved residues across species

  • Predicted transmembrane domains

  • Potential phosphorylation or glycosylation sites

  • Regions with predicted functional motifs

• Mutagenesis approach:

  • PCR-based site-directed mutagenesis

  • Gibson assembly for larger modifications

  • CRISPR-Cas9 for genomic modifications in cell lines

• Functional assessment:

  • Expression level comparison to wild-type

  • Subcellular localization analysis

  • Interaction partner binding assays

  • Functional activity measurements

• Structure-function correlation:

  • Mapping critical residues to predicted structural models

  • Comparing effects of conservative versus non-conservative substitutions

  • Evaluating the impact of mutations on protein stability and folding

What computational approaches are valuable for predicting TMEM183 structure and interactions?

Advanced computational methods for TMEM183 analysis include:

• Structure prediction:

  • Transmembrane topology prediction (TMHMM, Phobius)

  • Homology modeling using related proteins

  • Ab initio modeling for unique regions

  • AlphaFold2 or RoseTTAFold for whole protein prediction

• Interaction prediction:

  • Molecular docking simulations

  • Protein-protein interaction network analysis

  • Coevolution analysis for predicting interaction interfaces

  • Molecular dynamics simulations of membrane integration

• Functional prediction:

  • Conserved domain identification

  • Functional motif recognition

  • Gene ontology term assignment

  • Integration with tissue-specific expression data

These computational approaches can guide experimental design and help interpret experimental results within a broader structural and functional context.

How can recombinant TMEM183 be incorporated into proteomics workflows for identifying interaction partners?

Incorporating TMEM183 into proteomics workflows requires:

• Bait preparation strategies:

  • Expression with affinity tags (His, GST, FLAG)

  • Validation of functional integrity after tagging

  • Immobilization on appropriate matrices

• Pull-down methodologies:

  • Co-immunoprecipitation with anti-TMEM183 antibodies

  • Tandem affinity purification for higher purity

  • Proximity-based labeling (BioID, APEX) for transient interactions

  • Crosslinking approaches for capturing weak interactions

• Mass spectrometry analysis:

  • In-gel or in-solution digestion

  • LC-MS/MS analysis using parameters similar to:

    • Parent ion mass tolerance: 10 ppm

    • MS/MS ion mass tolerance: 0.02 Da

    • Appropriate modification considerations

• Data analysis:

  • Comparison against negative controls

  • Statistical filtering for significant interactions

  • Network analysis of identified partners

  • Validation of key interactions through orthogonal methods