Recombinant Human Transmembrane protein 191B (TMEM191B)

What is TMEM191B and why is it significant for research?

TMEM191B is a human transmembrane protein that belongs to the broader family of membrane-spanning proteins involved in various cellular processes. Like other transmembrane proteins, TMEM191B contains domains that traverse the cell membrane, potentially playing roles in signal transduction, molecular transport, or structural organization. The significance of TMEM191B in research stems from its potential involvement in cellular communication pathways and membrane organization. Understanding its structure and function contributes to our knowledge of membrane biology and potential therapeutic applications. When studying TMEM191B, researchers typically employ recombinant expression systems to produce sufficient quantities for functional and structural analyses.

Which expression systems are most effective for recombinant TMEM191B production?

Mammalian expression systems are generally the most effective for recombinant TMEM191B production due to their ability to properly fold complex transmembrane proteins and add appropriate post-translational modifications. Based on studies with similar transmembrane proteins, HEK293 cell lines (particularly HEK293S-TetR and HEK293S-GnTI-) have demonstrated superior expression capabilities for membrane proteins . For inducible expression systems, the T-REx 293 cell line with CMV/TetO2 promoter (using pcDNA4 vector systems) has shown excellent results for related transmembrane proteins, with expression levels reaching 1.22-1.67 million copies per cell for similar proteins .

The table below summarizes effective expression systems for membrane proteins similar to TMEM191B:

What purification strategies yield the highest purity of functional TMEM191B?

Purification of functional TMEM191B requires careful consideration of detergent selection and stabilization conditions. For transmembrane proteins, a multi-step purification approach typically yields the best results. Initially, cell membrane preparation through differential centrifugation should be performed, followed by solubilization using mild detergents such as DDM (n-dodecyl-β-D-maltopyranoside) or LMNG (lauryl maltose neopentyl glycol) that maintain protein structure and function.

Affinity chromatography using tags like His6 or FLAG is recommended as the first purification step, followed by size exclusion chromatography (SEC) to separate protein aggregates and obtain homogeneous preparations. Throughout the purification process, it is crucial to maintain cholesterol levels in the buffer, as membrane proteins like TMEM191B often have strict requirements for cholesterol to stabilize their folding state . Removal of cholesterol can abolish functional activity, which can be recovered upon addition of exogenous cholesterol but not with cholesterol analogues .

How can researchers optimize TMEM191B expression in mammalian cell systems?

Optimizing TMEM191B expression in mammalian cell systems requires a multifaceted approach addressing gene design, expression conditions, and cellular machinery. Based on experience with similar transmembrane proteins, researchers should consider the following optimization strategies:

• Codon optimization: Adjust the coding sequence to match the codon bias of the host cell line while avoiding rare codons that could impede translation efficiency.

• Signal sequence modification: N-terminal signal sequences significantly impact membrane protein trafficking. For TMEM191B, testing multiple signal sequences (e.g., native vs. heterologous) can identify optimal trafficking patterns.

• Induction parameters: For inducible systems, carefully titrate inducer concentration (e.g., tetracycline) and induction timing. Extended induction periods at lower temperatures (30-32°C) often improve proper folding of complex transmembrane proteins .

• Culture supplementation: Add specific lipids, particularly cholesterol, which has been shown to critically stabilize the folding state of transmembrane proteins. Research indicates that removal of cholesterol abolishes transport activity and inhibitor binding, which can be recovered upon addition of exogenous cholesterol .

• Molecular chaperone co-expression: Co-expression of chaperones like calnexin, which assists with proper folding, particularly for N-glycosylated proteins. Studies with other transporters have demonstrated that calnexin is required for efficient folding of membrane proteins in the endoplasmic reticulum .

The expression optimization process should follow a systematic experimental design with proper controls, as outlined in sections 3.1 and 3.2.

What are the most reliable methods for assessing TMEM191B functionality after purification?

Assessing TMEM191B functionality after purification requires a combination of biophysical and biochemical approaches. Techniques should be selected based on the hypothesized function of TMEM191B and its potential interaction partners:

• Ligand binding assays: If TMEM191B is involved in molecular recognition or transport, radioligand binding assays or fluorescence-based binding assays can quantify binding affinities and kinetics. These should be performed with purified TMEM191B reconstituted into lipid bilayers or nanodiscs to maintain native conformation.

• Microscale thermophoresis (MST): This technique can measure interactions between purified TMEM191B and potential binding partners with minimal protein consumption, providing affinity constants (KD values) with high sensitivity.

• Proteoliposome-based functional assays: Reconstituting TMEM191B into liposomes allows for transport or channel activity measurements, particularly if the protein functions as a transporter or ion channel.

• Circular dichroism (CD) spectroscopy: This technique provides information about secondary structure integrity and can be used to compare wild-type TMEM191B with mutants or to assess structural changes upon ligand binding.

• Thermal stability assays: Techniques like differential scanning fluorimetry can evaluate the stability of the purified protein under various conditions, helping identify stabilizing factors for structural and functional studies.

For transmembrane proteins like TMEM191B, maintaining the proper lipid environment is crucial for functional assessment. Research has shown that cholesterol is particularly important for maintaining the functional state of membrane transporters, with activity being abolished upon cholesterol removal .

How do post-translational modifications impact TMEM191B structure and function?

Post-translational modifications (PTMs) significantly impact TMEM191B structure, localization, and functional properties. N-glycosylation is particularly important for transmembrane proteins, affecting both folding efficiency and quality control during biosynthesis:

• N-glycosylation: Based on studies of similar transporters, N-glycosylation likely impacts TMEM191B folding and quality control rather than directly affecting function. Research on similar transporters like SERT has shown that while removal of N-glycosylation sites reduced surface expression by 50%, the transport kinetics (Km) and inhibitor binding (KD) remained unaffected . This suggests that N-glycosylation primarily influences protein biogenesis and trafficking rather than the fundamental transport mechanism.

• Calnexin interaction: The molecular chaperone calnexin likely interacts with glycosylated forms of TMEM191B as part of the quality control system in the endoplasmic reticulum. This interaction is critical for efficient folding of transmembrane proteins, and is directly related to the N-glycosylation status as calnexin binds specifically to glucosylated forms of N-glycans .

• Cholesterol dependence: While not a traditional PTM, cholesterol association with TMEM191B likely stabilizes the protein's folding state. Studies on similar transporters have demonstrated a strict requirement for cholesterol, with transport activity and inhibitor binding being abolished upon cholesterol removal . This effect is specific to cholesterol, as it cannot be recovered with cholesterol analogues.

What experimental design approach is most appropriate for studying TMEM191B function?

When designing experiments to study TMEM191B function, researchers should implement a rigorous experimental design framework that accounts for the specific challenges of membrane protein research. Based on established experimental design principles, the following approach is recommended:

• Hypothesis formulation: Begin with a clearly defined research question about TMEM191B function, such as its potential transport activity, interaction partners, or structural characteristics. The relationship between variables in your study will determine your experimental approach .

• Variable definition: Define the independent variable (the intended stimuli, such as ligand concentration or mutation) and the dependent variable (the expected effect, such as binding affinity or structural change). After identifying these variables, consider how to control them in your experiment .

• Control implementation: For transmembrane protein studies, multiple controls are essential:

  • Negative controls (empty vector or inactive mutant)

  • Positive controls (well-characterized related transmembrane protein)

  • Vehicle controls for all reagents

• True experimental design implementation: Whenever possible, implement a true experimental design with randomization and proper controls. The "Pretest-Posttest Control Group Design" is particularly effective for TMEM191B studies, where baseline measurements are established before introducing variables .

• Bayesian optimal experimental design: For complex studies involving multiple variables, consider implementing Bayesian optimal experimental design (BOED) approaches that can maximize information gain while minimizing experimental resources. This approach is particularly valuable when optimizing expression conditions or structural studies .

When studying TMEM191B, focus on designing experiments that maximize expected information gain (EIG) while accounting for the practical constraints of membrane protein research . This approach ensures efficient use of resources while generating robust and reproducible data.

What controls should be included when studying recombinant TMEM191B expression?

Robust controls are essential when studying recombinant TMEM191B expression to ensure reliable and interpretable results. The following controls should be implemented:

• Expression vector controls:

  • Empty vector control (transfection without TMEM191B gene)

  • GFP or other reporter control (to assess transfection efficiency)

  • Known membrane protein control (to validate the expression system)

• Cell line controls:

  • Untransfected cell control

  • Cell viability assessments before and after induction

  • Comparison of multiple cell lines (e.g., HEK293T vs. T-REx 293) to identify optimal expression systems

• Expression condition controls:

  • Temperature variation (30°C, 33°C, 37°C)

  • Induction timing series (24h, 48h, 72h)

  • Inducer concentration gradient

• Protein quality controls:

  • Size exclusion chromatography to assess aggregation state

  • Circular dichroism to evaluate secondary structure integrity

  • Thermal stability measurements under various buffer conditions

• Functional controls:

  • Known functional mutants (if available)

  • Detergent/lipid variation to assess functional sensitivity

  • Cholesterol depletion and repletion tests, which have been shown to be critical for transmembrane protein function

How can researchers troubleshoot low expression or misfolding of recombinant TMEM191B?

Troubleshooting low expression or misfolding of recombinant TMEM191B requires a systematic approach addressing multiple potential issues:

• Gene sequence optimization:

  • Check for rare codons in the TMEM191B sequence

  • Verify the absence of cryptic splice sites

  • Ensure the absence of internal Shine-Dalgarno-like sequences that could cause translational pausing

• Expression vector elements:

  • Evaluate promoter strength (CMV promoters typically provide strong expression in mammalian cells)

  • Test different signal sequences for improved targeting

  • Consider using an inducible system like CMV/TetO2 in T-REx 293 cells, which has shown success with similar membrane proteins

• Cell line selection and culture conditions:

  • Compare expression in multiple cell lines (HEK293 variants have shown superior results for membrane proteins)

  • Test expression at reduced temperatures (30-32°C), which often improves folding

  • Supplement growth media with specific lipids, particularly cholesterol, which is critical for membrane protein folding and stability

• Co-expression strategies:

  • Co-express molecular chaperones (e.g., calnexin) that assist with membrane protein folding

  • Test co-expression with protein disulfide isomerase for proteins with multiple disulfide bonds

  • Consider fusion partners that enhance expression or folding

• Post-translation modification assessment:

  • Verify N-glycosylation status, as this affects membrane protein trafficking

  • Test expression in glycosylation-deficient cell lines (e.g., HEK293S-GnTI-) for structural studies

  • Evaluate the role of cholesterol in stabilizing TMEM191B folding

When implementing these troubleshooting strategies, follow a systematic experimental design approach, testing one variable at a time while maintaining appropriate controls . Document all conditions and results in a structured format to identify patterns and optimal conditions.

What cell lines are optimal for functional studies of TMEM191B?

The selection of appropriate cell lines is critical for functional studies of TMEM191B. Based on extensive data with similar membrane proteins, the following cell lines are recommended:

• HEK293 variant cell lines:

  • T-REx 293: This tetracycline-inducible cell line has demonstrated excellent results for membrane proteins, achieving expression levels of 400,000 copies per cell for serotonin transporters and up to 1.67 million copies per cell in optimized conditions . The inducible nature allows for precise control over expression timing.

  • HEK293S-TetR: Another tetracycline-inducible line that has shown high expression levels of up to 5.5 million copies per cell for membrane proteins like bradykinin receptors . This line is particularly useful for proteins that may be toxic when constitutively expressed.

  • HEK293S-GnTI-: This glycosylation-deficient cell line produces proteins with homogeneous N-glycans, which is advantageous for structural studies and has been successfully used in bioreactor cultures to achieve high cell densities (9.6 million cells/ml) .

• BHK-21 cells:

  • These cells have been successfully used with Semliki Forest Virus expression systems, achieving high expression levels for membrane proteins (up to 285 pmol/mg for adenosine receptors) .

• Flp-In T-REx 293 cells:

  • This system combines site-specific integration with inducible expression, providing more consistent expression levels between experiments and reducing position effects .

When selecting a cell line for TMEM191B studies, consider the intended application. For pure protein production, HEK293S-GnTI- in bioreactor culture has demonstrated high yields. For functional studies, T-REx 293 offers a good balance of expression level and native post-translational modifications. Regardless of the cell line selected, cholesterol maintenance is critical, as research has demonstrated that membrane proteins have strict requirements for cholesterol to maintain their folded, functional state .

What are the recommended approaches for scaling up TMEM191B production?

Scaling up TMEM191B production requires strategic approaches that balance yield, quality, and resources. Based on successful scale-up methods for similar membrane proteins, the following approaches are recommended:

• Suspension culture systems:

  • Transition adherent cells to suspension adaptation using specialized media

  • BHK-21 cells have been successfully used in suspension culture at 0.5 million cells/ml for membrane protein expression

  • MEL cells can be grown in suspension at 1 million cells/ml for similar applications

• Bioreactor implementation:

  • HEK293S-GnTI- cells have been successfully cultured in bioreactors at densities of 9.6 million cells/ml, yielding 3 mg of purified membrane protein per liter of culture

  • 3-5.5 liter bioreactor setups have produced 6-9 mg/l of membrane proteins like rhodopsin

  • Bioreactors provide superior control over pH, dissolved oxygen, and nutrient levels

• Production optimization strategies:

  • Implement fed-batch processes to extend culture viability

  • Optimize induction timing based on growth curve analysis

  • Consider reduced temperature cultivation (30-32°C) during protein expression phase

• Alternative scale-up methods:

  • CellStack systems have been successfully used for chemokine receptor production

  • Spinner flask cultivation provides an intermediate step between tissue culture and bioreactors

  • For initial studies, scaling with multiple tissue culture plates remains viable (yields of approximately 12-30 μg of purified protein per 15 cm plate have been reported)

The table below summarizes scale-up approaches and expected yields for membrane proteins similar to TMEM191B:

How should researchers analyze and interpret TMEM191B structural data?

Analyzing and interpreting structural data for TMEM191B requires specialized approaches that account for the unique challenges of membrane protein structural biology. Researchers should consider the following methodological framework:

• Homology modeling preliminary assessment:

  • Begin with sequence alignment against similar transmembrane proteins with known structures

  • Generate homology models using multiple templates to identify conserved structural features

  • Validate models through energy minimization and Ramachandran plot analysis

• Experimental structural data collection:

  • X-ray crystallography: Requires highly pure, homogeneous, and stable protein preparations, often facilitated by using HEK293S-GnTI- cells to produce protein with homogeneous glycosylation

  • Cryo-electron microscopy: Particularly valuable for TMEM191B, as it requires less protein and can visualize the protein in a more native-like lipid environment

  • NMR spectroscopy: For examining specific domains or ligand interactions with isotopically labeled protein

• Data analysis protocol:

  • Implement rigorous statistical analysis following Bayesian optimal experimental design principles to maximize information gain from limited data

  • Compare multiple structural models and evaluate them against experimental data

  • Conduct molecular dynamics simulations to assess structural stability and flexibility in membrane environments

• Structure-function correlation:

  • Map functional data onto structural models to identify key regions

  • Evaluate the impact of cholesterol binding sites, as cholesterol has been shown to be critical for membrane protein stability and function

  • Assess the role of N-glycosylation sites in protein folding and trafficking, rather than direct functional impact

• Integration with complementary techniques:

  • Cross-linking mass spectrometry to validate protein-protein interactions

  • Hydrogen-deuterium exchange mass spectrometry to examine conformational dynamics

  • Site-directed mutagenesis combined with functional assays to validate structural hypotheses

When designing structural biology experiments for TMEM191B, researchers should follow a systematic experimental design approach that maximizes expected information gain while accounting for practical constraints . This ensures efficient use of resources while generating the most valuable structural insights.

What emerging technologies might advance TMEM191B research?

Emerging technologies offer promising opportunities to overcome current limitations in TMEM191B research:

• Advanced expression systems:

  • Cell-free protein synthesis systems are increasingly effective for membrane proteins, allowing rapid production without cell culture

  • Baculovirus-mammalian cell hybrid systems that combine high transduction efficiency with mammalian post-translational modifications

  • Gene editing approaches like CRISPR-Cas9 to create stable cell lines with optimized expression characteristics

• Cutting-edge structural biology techniques:

  • MicroED (micro-electron diffraction) for structural analysis of small crystals

  • Integrative structural biology approaches combining multiple experimental techniques

  • AI-powered structure prediction tools like AlphaFold2, which are increasingly accurate even for membrane proteins

• Spatial genomics and proteomics:

  • Implementation of spatial transcriptomics to understand TMEM191B expression patterns in tissues

  • Advanced experimental design approaches for spatial studies using Bayesian optimal experimental design (BOED) to maximize information gain

  • Single-cell technologies to understand cell-to-cell variability in TMEM191B expression and function

• Novel membrane mimetics:

  • Styrene maleic acid lipid particles (SMALPs) that extract membrane proteins with their native lipid environment

  • Designed amphipathic peptides that form membrane-mimetic environments

  • Lipid nanodiscs with defined composition for controlled functional studies

• Advanced functional characterization:

  • Label-free biosensors for real-time interaction studies

  • Single-molecule techniques to examine conformational dynamics

  • Optogenetic approaches to control TMEM191B function with light