Recombinant Human Transmembrane protein 99 (TMEM99)

What is the basic structure of human TMEM99?

Human TMEM99 (also known as MGC21518) is a transmembrane protein encoded by a gene located on chromosome 17q21.2 . The protein consists of 258 amino acids with the mature protein spanning residues 20-258 . TMEM99 has multiple transmembrane domains, as is characteristic of proteins in this class. The protein sequence contains several hydrophobic regions that span the membrane, with specific amino acid sequences that determine its topology and orientation within the cellular membrane.

The primary structure of TMEM99 contains multiple alpha-helical transmembrane domains, which is consistent with predictions for transmembrane proteins using hidden Markov model-based tools like TMHMM that can predict transmembrane helices with 97-98% accuracy . These transmembrane domains are primarily composed of hydrophobic amino acids that facilitate integration into the lipid bilayer.

What are the known transcript variants of TMEM99?

TMEM99 is known to have multiple transcript variants, with transcript variant 3 (NM_001195387) being well-characterized and available as an expression-ready ORF clone . This variant has an ORF size of 774 base pairs . The existence of multiple transcript variants suggests potential differential expression or function across various tissues or developmental stages.

Each transcript variant may result in slightly different protein isoforms, which could have distinct functional properties or subcellular localizations. Researchers should carefully select the appropriate variant for their specific experimental questions.

What is currently known about TMEM99's cellular localization and function?

While specific functional data on TMEM99 is limited in the provided search results, as a transmembrane protein, it is integrated into cellular membranes. The Allen Human Brain Atlas indicates TMEM99 gene expression in brain tissue , suggesting potential neurological functions.

Like many transmembrane proteins, TMEM99 likely plays roles in:

• Cellular signaling

• Transport of molecules across membranes

• Structural support for membrane organization

• Potential interactions with other membrane or cytosolic proteins

The absence of specific pathway information in search result (where pathway fields were empty) indicates that detailed functional characterization of TMEM99 may still be ongoing, presenting opportunities for novel research.

What expression systems are optimal for producing recombinant TMEM99?

Based on available information, recombinant TMEM99 can be produced in both prokaryotic (E. coli) and eukaryotic expression systems. For applications requiring post-translational modifications, mammalian expression systems are preferable, although TMEM99 may present expression challenges typical of transmembrane proteins .

When choosing an expression system, consider:

Selection markers for mammalian expression include Neomycin, while E. coli selection typically uses Ampicillin (100 μg/mL) .

What are the common challenges in expressing recombinant TMEM99 and how can they be addressed?

Transmembrane proteins like TMEM99 are often categorized as "difficult to express" proteins in recombinant systems due to several challenges:

• Bottlenecks in the protein expression pathway: Research has shown that limitations can occur at various stages of expression, from transcription to post-translational processing .

• Post-translational processing issues: After initial processing in the endoplasmic reticulum, transmembrane proteins may encounter difficulties in proper folding, membrane insertion, or trafficking .

• Problematic sequence features: Computational analyses indicate that increased abundance of positively-charged or hydrophobic surface regions correlates with poor protein secretion .

Methodological approaches to address these challenges include:

• Protein engineering: Modification of problematic sequence features, particularly positively-charged or hydrophobic surface regions that may impede proper expression .

• Expression vector optimization: Using vectors with strong promoters appropriate for the expression system.

• Culture condition optimization: Adjusting temperature, induction timing, and media composition to improve protein yield and solubility.

• Fusion tags: Incorporating solubility-enhancing tags like GST or MBP, or epitope tags for detection and purification.

What purification strategies are effective for recombinant TMEM99?

Purification of transmembrane proteins like TMEM99 requires specialized approaches:

• Affinity chromatography: The available recombinant TMEM99 contains a His-tag , enabling purification via immobilized metal affinity chromatography (IMAC). This approach allows:

  • Selective binding of His-tagged TMEM99

  • Washing away of contaminants

  • Elution with imidazole or low pH

• Detergent solubilization: Since TMEM99 is a membrane protein, effective purification requires:

  • Selection of appropriate detergents (e.g., DDM, CHAPS, or Triton X-100)

  • Optimization of detergent concentration to maintain protein solubility without denaturation

  • Careful buffer composition to preserve protein structure

• Size exclusion chromatography: As a polishing step to:

  • Remove aggregates

  • Ensure homogeneity

  • Exchange into final buffer conditions

Researchers should validate protein purity and structure after purification using techniques such as SDS-PAGE, Western blotting, and circular dichroism to ensure proper folding.

How can structural studies of TMEM99 be approached?

Structural characterization of transmembrane proteins like TMEM99 presents unique challenges but can be addressed through multiple complementary approaches:

• X-ray crystallography: Requires:

  • High-purity, homogeneous protein preparations

  • Crystallization trials with various detergents and lipidic conditions

  • Screening of crystallization conditions (temperature, pH, additives)

  • Consideration of lipidic cubic phase crystallization for membrane proteins

• Cryo-electron microscopy (cryo-EM):

  • Increasingly powerful for membrane protein structure determination

  • May not require crystallization

  • Can visualize proteins in different conformational states

  • Requires optimization of grid preparation and detergent concentration

• NMR spectroscopy:

  • Suitable for smaller domains or transmembrane segments

  • Provides dynamic information

  • Requires isotopic labeling (15N, 13C)

  • Often combined with detergent micelles or bicelles

• Computational structure prediction:

  • Hidden Markov models can predict transmembrane topology with 97-98% accuracy

  • Modern AI-based methods (AlphaFold2, RoseTTAFold) show promise for membrane protein prediction

  • Should be validated with experimental data

What is known about TMEM99's potential role in protein aggregation or amyloid formation?

While direct evidence for TMEM99's role in amyloid formation is not presented in the search results, research has shown that α-helical transmembrane proteins can form amyloid-like fibrils under destabilizing conditions . This suggests several research directions:

• Investigation of TMEM99 stability:

  • Thermal stability assays (differential scanning fluorimetry)

  • Chemical denaturation studies

  • Assessment of aggregation propensity under varying pH and salt conditions

• Amyloid formation potential:

  • Identification of amyloidogenic sequences within TMEM99 using prediction algorithms

  • Testing for fibril formation using Thioflavin T fluorescence assays

  • Electron microscopy analysis of potential fibrils

• Relationship to disease mechanisms:

  • Assessment of TMEM99 in neurodegenerative disease models, given its brain expression

  • Potential interactions with known amyloidogenic proteins

  • Influence of disease-associated mutations on stability and aggregation

This research direction is particularly relevant given the expression of TMEM99 in brain tissue and the association of protein aggregation with numerous neurological disorders.

How might TMEM99 expression patterns in brain tissue be analyzed?

Given the evidence for TMEM99 expression in brain tissue from the Allen Human Brain Atlas , researchers might pursue several approaches to characterize its neuroanatomical distribution and function:

• Analysis of publicly available brain transcriptome data:

  • Allen Human Brain Atlas data mining

  • Comparison with Neurosynth term maps for functional correlations

  • Analysis across different brain regions and developmental stages

• Experimental visualization techniques:

  • Immunohistochemistry using anti-TMEM99 antibodies

  • In situ hybridization for mRNA localization

  • Single-cell RNA sequencing to identify specific neuronal or glial populations expressing TMEM99

• Functional significance assessment:

  • Correlation with brain region-specific functions

  • Comparison with expression patterns of interacting proteins

  • Investigation in neurodevelopmental or neurodegenerative disease models

• Gene expression studies:

  • qPCR analysis across brain regions

  • Western blotting for protein levels

  • Promoter analysis to understand regulation mechanisms

What are common issues in detecting TMEM99 in experimental systems?

Detection of transmembrane proteins like TMEM99 presents several technical challenges:

• Antibody accessibility issues:

  • Epitopes may be masked within the membrane

  • Conformational dependence of antibody recognition

  • Solution: Use multiple antibodies targeting different regions or tagged recombinant constructs

• Western blot challenges:

  • Incomplete transfer of hydrophobic proteins

  • Protein aggregation during sample preparation

  • Solution: Optimize detergent conditions, transfer parameters, and consider specialized transfer methods for hydrophobic proteins

• Low abundance issues:

  • Natural expression levels may be low

  • Solution: Use enrichment techniques, sensitive detection methods, or overexpression systems

• Fluorescent protein fusion considerations:

  • Available GFP-tagged constructs (pCMV6-AC-GFP) can aid detection

  • Verify that tags don't interfere with localization or function

  • Consider tag position (N- or C-terminal) based on predicted topology

How can protein-protein interactions of TMEM99 be effectively studied?

Understanding TMEM99's interactions with other proteins requires specialized approaches for membrane proteins:

• Co-immunoprecipitation adaptations:

  • Use of appropriate detergents to solubilize membrane complexes without disrupting interactions

  • Cross-linking approaches to stabilize transient interactions

  • Stringent controls to avoid non-specific interactions with detergent micelles

• Proximity labeling approaches:

  • BioID or APEX2 fusion constructs to label proximal proteins in living cells

  • TurboID for rapid labeling kinetics

  • Spatial restriction of labeling enzymes to specific membrane compartments

• Fluorescence-based interaction methods:

  • Förster Resonance Energy Transfer (FRET) with fluorescent protein-tagged constructs

  • Bimolecular Fluorescence Complementation (BiFC)

  • Fluorescence Correlation Spectroscopy (FCS) for dynamic interactions

• Advanced mass spectrometry approaches:

  • Crosslinking Mass Spectrometry (XL-MS)

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

  • Label-free quantitative proteomics with appropriate controls

What experimental designs can assess the functional significance of TMEM99?

To elucidate TMEM99's functional roles, several experimental approaches can be employed:

• Gene editing strategies:

  • CRISPR-Cas9 knockout or knockdown

  • Site-directed mutagenesis of key residues

  • Domain deletion or swapping

• Overexpression studies:

  • Utilize available expression constructs like pCMV6-AC-GFP

  • Assess phenotypic changes in relevant cell types

  • Combine with rescue experiments in knockout models

• Localization and trafficking analysis:

  • Live-cell imaging of GFP-tagged TMEM99

  • Colocalization with organelle markers

  • Photoactivatable or photoconvertible tags for dynamic studies

• Functional assays based on predicted roles:

  • If transport function is suspected: substrate transport assays

  • If signaling role is predicted: pathway activation measurements

  • If structural role is hypothesized: membrane organization studies

• Brain-specific functional investigations:

  • Given brain expression , consider neuron-specific phenotypes

  • Electrophysiological measurements in expression systems

  • Neurite outgrowth, synapse formation, or other neuronal function assays

What emerging technologies might advance TMEM99 research?

Several cutting-edge approaches could significantly enhance our understanding of TMEM99:

• Single-particle cryo-EM advancements:

  • Improved detectors and processing algorithms

  • Smaller protein size limitations

  • Better resolution of flexible regions

• Integrative structural biology:

  • Combining multiple structural techniques (X-ray, NMR, cryo-EM)

  • Computational modeling with experimental constraints

  • Molecular dynamics simulations in membrane environments

• Single-molecule techniques:

  • Force spectroscopy for conformational studies

  • Single-molecule FRET for dynamic analysis

  • High-speed AFM for visualizing conformational changes

• Advanced genetic approaches:

  • Base editing for precise mutagenesis

  • Conditional knockouts for tissue-specific studies

  • CRISPR activation/interference for expression modulation

• AI-enhanced functional prediction:

  • Machine learning to identify functional motifs

  • Deep learning models for interaction network prediction

  • Structure-based function prediction

How might understanding TMEM99 contribute to broader transmembrane protein research?

TMEM99 research can provide valuable insights into general principles of membrane protein biology:

• Expression and folding mechanisms:

  • Overcoming challenges in recombinant membrane protein production

  • Understanding sequence determinants of membrane protein folding

  • Developing improved methods for membrane protein expression

• Structure-function relationships:

  • Correlation between transmembrane topology and function

  • Identification of conserved functional motifs

  • Understanding dynamics in the membrane environment

• Disease implications:

  • Given its brain expression , potential roles in neurological disorders

  • Possible contributions to membrane protein aggregation disorders

  • Identification of novel therapeutic targets

• Evolutionary perspectives:

  • Comparison across species for functional conservation

  • Analysis of paralogous TMEM family proteins

  • Understanding selective pressures on membrane protein evolution