Recombinant Stomatin-2 (sto-2)

What is Stomatin-like protein 2 (STOML2) and what are its primary cellular locations?

Stomatin-like protein 2 (STOML2), also known as SLP-2, is a member of the Band 7/mec-2 protein family. In humans, the canonical protein consists of 356 amino acid residues with a molecular mass of approximately 38.5 kDa. STOML2 primarily localizes to three cellular compartments: the cell membrane, mitochondria, and cytoplasm. It is widely expressed at low levels across multiple tissues and is particularly important for regulating mitochondrial biogenesis and activity. The protein undergoes post-translational modifications, notably phosphorylation, which may regulate its function. Alternative splicing generates two different isoforms of this protein in humans, potentially contributing to tissue-specific functions .

Alternative nomenclature for this protein includes mitochondrial stomatin-like protein 2, EPB72-like 2, EPB72-like protein 2, paraprotein target 7, and HSPC108. Orthologous versions have been identified across multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, indicating its evolutionary conservation and biological significance .

What expression systems are most effective for producing recombinant Stomatin-2?

Based on successful protocols in the literature, bacterial expression systems, particularly E. coli BL21(DE3) strains, have proven effective for recombinant Stomatin-2 production. For optimal expression:

• Clone the full-length cDNA sequence into a vector containing an N-terminal hexahistidine tag (such as pDEST17).

• Transform the construct into BL21(DE3) E. coli cells.

• Grow transformed bacteria in LB medium supplemented with appropriate antibiotics to an OD600 of 0.6.

• Induce protein expression with 1 mM IPTG and incubate for 3-4 hours at 37°C.

• Harvest cells by centrifugation and resuspend in appropriate lysis buffer (e.g., 50 mM sodium phosphate pH 8.3, 0.3 M NaCl) .

This approach typically yields the protein in inclusion bodies, which requires subsequent refolding. For applications requiring native protein conformation, mammalian or insect cell expression systems may be preferable, though these weren't specifically described in the available literature for STOML2.

What are the most effective methods for purifying recombinant Stomatin-2?

Purification of recombinant Stomatin-2 typically involves a multi-step process:

• Cell lysis: Disrupt bacterial cells using a French press (1500 psi) or sonication in lysis buffer (50 mM sodium phosphate pH 8.3, 0.3 M NaCl).

• Inclusion body isolation: Centrifuge the lysate at 20,000 × g for 40 minutes to pellet inclusion bodies.

• Wash steps: Perform at least two washes with wash buffer containing detergents and mild denaturants (lysis buffer supplemented with 2% Triton X-100, 2 M urea).

• Solubilization: Resuspend the washed inclusion bodies in solubilization buffer containing strong denaturants (lysis buffer with 5 mM beta-mercaptoethanol, 20 mM imidazole, 8 M urea).

• Protein refolding: Dilute the solubilized protein 100-fold into equilibration buffer (without urea) to promote refolding.

• Affinity chromatography: Purify the refolded protein using Ni²⁺-NTA affinity chromatography under native conditions, with a 20-500 mM imidazole gradient for elution.

• Buffer exchange: Replace the elution buffer with an appropriate storage buffer (such as PBS pH 7.4) using dialysis or gel filtration .

This protocol yields purified recombinant Stomatin-2 suitable for most research applications, including antibody production and functional studies.

How can the identity and quality of purified recombinant Stomatin-2 be verified?

To ensure the identity and quality of purified recombinant Stomatin-2:

• SDS-PAGE analysis: Confirm expected molecular weight (approximately 38.5 kDa plus any tag contribution) and purity.

• Western blotting: Use commercially available anti-Stomatin-2 antibodies to verify protein identity.

• Mass spectrometry: Perform peptide mass fingerprinting or LC-MS/MS to confirm protein sequence and identify any post-translational modifications.

• Size exclusion chromatography: Assess protein homogeneity and detect potential aggregation.

• Circular dichroism: Evaluate proper protein folding by analyzing secondary structure elements.

• Functional assays: Where possible, confirm bioactivity through appropriate functional tests related to mitochondrial function or protein-protein interactions.

For research requiring high protein quality, additional quality controls may include endotoxin testing (particularly important for immunological experiments) and thermal shift assays to assess protein stability.

What are the considerations for generating and validating antibodies against Stomatin-2?

Generating reliable antibodies against Stomatin-2 requires careful planning and validation:

Production Approach:

• Immunize animals (typically rodents) with purified recombinant Stomatin-2 using an appropriate adjuvant for the initial dose (such as TiterMax) followed by protein in PBS for subsequent boosts.

• Establish an immunization schedule with 21-day intervals between doses, administering approximately 100 μg of protein per dose.

• Collect serum 15 days after the final immunization .

Validation Strategy:

• Determine antibody titer and specificity using ELISA against the recombinant protein.

• Confirm reactivity by Western blot, starting with dilutions around 1:10,000. Verify that the antibody recognizes both recombinant protein and endogenous Stomatin-2 in relevant cell/tissue lysates.

• Perform immunofluorescence (dilutions around 1:100) to confirm subcellular localization patterns match known distributions (mitochondria, cell membrane, cytoplasm).

• Include appropriate negative controls such as pre-immune serum and positive controls like commercially validated antibodies.

• Conduct cross-reactivity tests against related stomatin family proteins to ensure specificity.

• If possible, validate antibody specificity using cells with STOML2 knockout or knockdown .

These comprehensive validation steps ensure that antibodies will be reliable tools for subsequent research applications.

How can recombinant Stomatin-2 be used to study protein-protein interactions?

Several methodologies can be employed to investigate Stomatin-2 interactions:

• Pull-down assays: Use His-tagged recombinant Stomatin-2 as bait with cell lysates, followed by identification of binding partners via mass spectrometry.

• Co-immunoprecipitation: Perform reciprocal co-IPs with antibodies against Stomatin-2 and suspected interaction partners, particularly focusing on mitochondrial proteins.

• Proximity labeling methods:

  • BioID approach: Create a Stomatin-2-BirA fusion protein to biotinylate proximal proteins in living cells

  • APEX2 system: Generate Stomatin-2-APEX2 fusions for spatially restricted labeling within mitochondria

• FRET/BRET analyses: Develop fluorescent protein fusions for live-cell interaction studies, particularly useful for dynamic interactions.

• Yeast two-hybrid screening: Use Stomatin-2 as bait to screen cDNA libraries, though this approach may be limited for mitochondrial proteins.

• Surface plasmon resonance: Measure direct binding kinetics between purified recombinant Stomatin-2 and candidate interacting proteins.

Since Stomatin-2 likely functions in regulating mitochondrial biogenesis and activity, particular attention should be paid to interactions with components of mitochondrial membranes and the mitochondrial protein synthesis machinery .

What approaches are effective for studying the role of Stomatin-2 in mitochondrial function?

To investigate Stomatin-2's role in mitochondrial function:

• Mitochondrial isolation and fractionation:

  • Isolate intact mitochondria using differential centrifugation

  • Perform sub-mitochondrial fractionation to determine precise localization (outer membrane, inner membrane, intermembrane space, matrix)

  • Use protease protection assays to determine topology

• Functional assays:

  • Measure oxygen consumption rate (OCR) using Seahorse XF analyzers in cells with Stomatin-2 knockout/knockdown

  • Assess mitochondrial membrane potential using fluorescent indicators (TMRM, JC-1)

  • Quantify ATP production rates and compare between control and Stomatin-2-deficient cells

  • Evaluate mitochondrial mass and morphology using MitoTracker dyes and confocal microscopy

• Proteomics approaches:

  • Perform differential proteomics on mitochondria from control vs. Stomatin-2-deficient cells

  • Use SILAC or TMT labeling for quantitative comparisons

  • Analyze changes in mitochondrial protein complexes using blue native PAGE

• Genetic models:

  • Generate Stomatin-2 knockout cell lines using CRISPR/Cas9

  • Create conditional knockout animal models to study tissue-specific effects

  • Develop fluorescently tagged Stomatin-2 for live-cell imaging of mitochondrial dynamics

Since Stomatin-2 is reported to regulate mitochondrial biogenesis and activity , these approaches will help elucidate its specific mechanistic roles within the organelle.

How can post-translational modifications of Stomatin-2 be effectively studied?

Post-translational modifications (PTMs) of Stomatin-2, particularly phosphorylation , can be studied using these approaches:

• Mass spectrometry-based PTM mapping:

  • Enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO₂)

  • Perform LC-MS/MS analysis using collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation

  • Quantify modification stoichiometry using targeted MS approaches

• Site-directed mutagenesis:

  • Generate phosphomimetic (S/T to D/E) and phosphodeficient (S/T to A) mutants

  • Express mutants in appropriate cell models and assess functional consequences

  • Compare subcellular localization patterns between wildtype and mutant proteins

• Specific PTM antibodies:

  • Develop modification-specific antibodies if key sites are identified

  • Use these for Western blotting and immunofluorescence to monitor modification dynamics

• In vitro kinase/phosphatase assays:

  • Identify enzymes responsible for Stomatin-2 phosphorylation/dephosphorylation

  • Perform in vitro assays with purified components to confirm direct modification

• Dynamic PTM profiling:

  • Monitor PTM changes during mitochondrial stress, cell cycle progression, or metabolic challenges

  • Combine with functional assays to correlate modifications with activity

This comprehensive approach will help uncover how PTMs regulate Stomatin-2's function in mitochondrial biology and potentially in other cellular compartments.

What immunological responses are triggered by recombinant Stomatin-2 in research models?

Studies with recombinant Stomatin-like proteins suggest complex immunological responses:

• Cytokine profile:

  • Recombinant Stomatin-like proteins can elicit a predominantly Th1-type immune response

  • Significant production of IFN-γ and TNF-α (pro-inflammatory cytokines)

  • Elevated IL-10 (immunomodulatory cytokine) levels

  • Minimal or undetectable IL-4 (Th2 cytokine) production

• Immunization protocols:

  • Multiple immunizations (typically 3-4 doses at 21-day intervals)

  • Initial dose formulated with complete Freund's adjuvant (CFA)

  • Subsequent doses often use incomplete Freund's adjuvant (IFA) or PBS

• Cellular responses:

  • Activation of splenocytes when re-stimulated with the recombinant protein

  • Specific T-cell proliferation in response to antigen

  • When studying immunological effects, consider including polymyxin B in culture systems to neutralize potential LPS contamination

These immunological properties suggest that recombinant Stomatin-2 may have applications beyond basic research, potentially serving as an immunogen in vaccine development strategies, though human applications would require alternative adjuvant formulations suitable for clinical use .

What are common challenges in recombinant Stomatin-2 expression and how can they be addressed?

Common challenges and their solutions include:

• Inclusion body formation:

  • Lower induction temperature (16-25°C instead of 37°C)

  • Reduce IPTG concentration (0.1-0.5 mM)

  • Consider fusion partners like MBP, SUMO, or thioredoxin to enhance solubility

  • Use specialized E. coli strains designed for improved protein folding

• Poor refolding efficiency:

  • Optimize refolding conditions by screening different pH values, salt concentrations, and additives

  • Try step-wise dialysis with gradually decreasing denaturant concentrations

  • Include appropriate redox systems (GSH/GSSG) to facilitate correct disulfide bond formation

  • Consider on-column refolding during affinity purification

• Protein aggregation after purification:

  • Include low concentrations of stabilizing agents (5% glycerol, 0.1% Triton X-100)

  • Optimize protein concentration and storage buffer composition

  • Store at appropriate temperature (typically -80°C in small aliquots)

• Low protein yield:

  • Optimize codon usage for E. coli expression

  • Consider auto-induction media instead of IPTG induction

  • Scale up culture volume while maintaining optimal growth conditions

  • Evaluate different expression vectors with stronger/weaker promoters

These adjustments can significantly improve the quality and yield of recombinant Stomatin-2 production for research applications.

How can recombinant Stomatin-2 be used to develop quantitative assays for research and diagnostics?

Recombinant Stomatin-2 can be leveraged to develop several quantitative assay formats:

• ELISA development:

  • Use purified recombinant Stomatin-2 to generate standard curves

  • Develop sandwich ELISA using two different antibodies recognizing distinct epitopes

  • Optimize coating, blocking, and detection conditions for maximum sensitivity and specificity

  • Validate assay parameters including limit of detection, dynamic range, precision, and accuracy

• Western blot quantification:

  • Create recombinant Stomatin-2 standards of known concentrations

  • Develop densitometric analysis protocols for relative quantification

  • Validate linearity of signal across relevant concentration ranges

• Flow cytometry applications:

  • Label recombinant Stomatin-2 with fluorescent dyes for binding studies

  • Develop intracellular staining protocols to quantify endogenous Stomatin-2 levels

  • Create calibration standards for quantitative flow cytometry

• Activity-based assays:

  • Develop functional assays based on known biochemical activities of Stomatin-2

  • Create high-throughput screening formats to identify modulators of Stomatin-2 function

When developing these assays, it's important to include proper controls and validation steps to ensure specificity for Stomatin-2 rather than related family members.

What emerging technologies might advance Stomatin-2 research?

Several emerging technologies hold promise for advancing Stomatin-2 research:

• Cryo-electron microscopy (Cryo-EM):

  • Determine high-resolution structures of Stomatin-2 in isolation and within protein complexes

  • Visualize Stomatin-2's interaction with mitochondrial membranes

• Single-cell technologies:

  • Analyze cell-to-cell variation in Stomatin-2 expression and localization

  • Correlate Stomatin-2 levels with mitochondrial parameters at single-cell resolution

• Genome editing advancements:

  • Prime editing and base editing for precise modification of Stomatin-2

  • Knockin of endogenous tags for studying native protein dynamics

• Spatial proteomics:

  • Map Stomatin-2's precise localization within mitochondrial subdomains

  • Identify spatial organization of Stomatin-2-containing complexes

• Optogenetic and chemogenetic tools:

  • Develop tools for acute control of Stomatin-2 activity or localization

  • Study temporal aspects of Stomatin-2 function in mitochondrial regulation

• Computational biology approaches:

  • Molecular dynamics simulations to understand Stomatin-2's membrane interactions

  • Machine learning models to predict functional consequences of Stomatin-2 variants

These technologies will enable researchers to address fundamental questions about Stomatin-2's structure, dynamics, and functional roles in health and disease.

How can contradictory findings in Stomatin-2 research be reconciled through experimental design?

Resolving contradictory findings in Stomatin-2 research requires systematic approaches:

• Standardized experimental systems:

  • Establish consensus cell lines and experimental conditions

  • Develop common protocols for protein production, purification, and functional assays

  • Create shared resources such as validated antibodies and expression constructs

• Comprehensive controls:

  • Include genetic knockouts as definitive negative controls

  • Use multiple independent methods to confirm key findings

  • Establish dose-response relationships rather than single-point measurements

• Context-dependent function analysis:

  • Systematically evaluate Stomatin-2 function across different cell types

  • Assess function under various metabolic states and stress conditions

  • Consider tissue-specific interaction partners

• Isoform-specific investigations:

  • Clearly distinguish between the two known isoforms of Stomatin-2

  • Develop isoform-specific reagents and assays

  • Investigate potential differential functions of each isoform

• Collaborative research initiatives:

  • Establish multi-laboratory validation studies for key findings

  • Create open data repositories for sharing primary data

  • Develop consensus guidelines for experimental protocols