Recombinant Human Putative transmembrane protein C13orf44 (C13orf44)

What is Recombinant Human Putative transmembrane protein C13orf44 and how is it classified?

C13orf44, now commonly referred to as SMIM2 (Small Integral Membrane Protein 2), is a putative transmembrane protein predicted to function as an integral component of cellular membranes. The protein has been identified across multiple species with conserved domains suggesting evolutionary importance . Structurally, C13orf44/SMIM2 belongs to a class of small membrane proteins that typically contain one or more transmembrane domains with relatively small extracellular and intracellular portions.

When expressed recombinantly, the protein is typically tagged (often with a 6xHis tag at the C-terminus) to facilitate purification and detection in experimental systems. The human variant is expressed in mammalian cell systems to ensure proper post-translational modifications and folding essential for maintaining native protein conformation and function .

What expression systems are recommended for C13orf44/SMIM2 production?

Mammalian expression systems are strongly preferred for C13orf44/SMIM2 production due to the protein's transmembrane nature and potential requirements for proper post-translational modifications. Based on current research protocols, the following expression systems have demonstrated successful production:

For optimal results, the protein should be expressed with appropriate tagging strategies (typically C-terminal His tags) that minimize interference with membrane insertion while facilitating downstream purification. Expression verification should be performed via Western blotting with tag-specific or protein-specific antibodies prior to functional characterization .

What purification strategies are effective for recombinant C13orf44/SMIM2?

Purification of membrane proteins like C13orf44/SMIM2 requires specialized approaches to maintain protein stability and functionality. The recommended purification protocol follows a multi-step approach:

• Cell lysis using mild detergents (e.g., 1% DDM, 0.5% CHAPS) that effectively solubilize membranes while preserving protein structure

• Affinity chromatography utilizing the His-tag, with binding buffers containing reduced detergent concentrations

• Size exclusion chromatography to remove aggregates and obtain homogeneous protein preparations

• Quality control assessment via SDS-PAGE and Western blotting to confirm purity (≥95% as assessed by SDS-PAGE)

For experimental applications requiring higher purity, additional ion-exchange chromatography may be incorporated between steps 2 and 3. The purified protein should be stored in stabilizing buffer conditions that maintain its native conformation, typically with appropriate detergent concentrations above their critical micelle concentration.

How can experimental design be optimized for functional characterization of C13orf44/SMIM2?

Functional characterization of membrane proteins like C13orf44/SMIM2 requires thoughtful experimental design that controls for various factors affecting protein behavior. Based on established experimental design principles, researchers should implement the following strategies:

• Systematic variable manipulation: When investigating C13orf44/SMIM2 function, independently manipulate key variables (expression level, cellular localization, interacting partners) while controlling for confounding factors .

• Selection of appropriate dependent variables: Measure multiple outcomes (protein localization, downstream signaling effects, interaction profiles) to comprehensively characterize function.

• Control for extraneous variables: Account for factors like cell type-specific effects, tag interference, and expression level variation that might confound results .

• Experimental validation: Employ multiple complementary techniques to validate findings:

  Technique	Application	Data Output	Limitations
  Immunofluorescence	Localization	Subcellular distribution	Antibody specificity
  Co-immunoprecipitation	Protein interactions	Binding partners	Weak/transient interactions may be missed
  CRISPR-Cas9 knockout	Loss-of-function	Phenotypic changes	Compensatory mechanisms
  Mass spectrometry	Interactome analysis	Comprehensive binding partners	Complex data interpretation

• Hypothesis formulation: Develop clear null and alternate hypotheses about C13orf44/SMIM2 function based on bioinformatic predictions and preliminary data .

When designing these experiments, researchers should incorporate proper negative controls (e.g., empty vector transfections, non-specific antibodies) and positive controls (known membrane proteins with similar characteristics) to ensure result validity.

What strategies can resolve contradictory findings in C13orf44/SMIM2 research?

Resolving contradictory findings in C13orf44/SMIM2 research requires systematic analysis of methodological differences and careful experimental design. When faced with contradictory data, researchers should:

• Compare experimental conditions: Systematically analyze differences in expression systems, tags, purification methods, and functional assays that may explain divergent results.

• Control for protein conformation: Verify that the recombinant protein maintains proper folding and membrane insertion across different experimental systems using conformation-sensitive techniques like limited proteolysis or circular dichroism.

• Implement meta-analytical approaches: Synthesize findings across multiple studies, weighting results based on methodological rigor and sample size.

• Design reconciliation experiments: Develop experiments specifically aimed at testing competing hypotheses under standardized conditions that bridge methodological differences between contradictory studies.

• Consider context-dependent functions: Investigate whether C13orf44/SMIM2 exhibits different functions in different cellular contexts, which may explain apparently contradictory findings.

A structured approach using factorial experimental design allows systematic evaluation of how different variables interact to influence C13orf44/SMIM2 function, potentially explaining contradictory findings in the literature .

How can researchers effectively validate the biological activity of recombinant C13orf44/SMIM2?

Validating the biological activity of recombinant C13orf44/SMIM2 requires multi-faceted approaches that assess both structural integrity and functional activity:

• Structural validation:

  • Circular dichroism spectroscopy to confirm secondary structure content

  • Limited proteolysis to verify proper folding

  • Size exclusion chromatography to assess oligomeric state

• Functional validation:

  • Membrane integration assays to confirm proper insertion into lipid bilayers

  • Protein-protein interaction studies via pull-down assays or surface plasmon resonance

  • Cell-based assays measuring downstream signaling events

• Comparative analysis:

  • Functional comparison between recombinant protein and endogenously expressed C13orf44/SMIM2

  • Cross-species activity comparison to identify conserved functions

Validation experiments should include appropriate positive controls (e.g., well-characterized membrane proteins) and negative controls (e.g., denatured protein samples), with activity assessed across multiple independent protein preparations to ensure reproducibility. Researchers should verify activity using ELISA and cell culture validation as standard quality control measures .

What immunological techniques are most effective for studying C13orf44/SMIM2?

Immunological techniques provide powerful tools for C13orf44/SMIM2 research, particularly when optimized for membrane protein analysis:

• Antibody selection considerations:

  • Epitope location relative to transmembrane domains

  • Cross-reactivity with related proteins

  • Performance in various applications (WB, IP, IF, FACS)

• Optimized immunoprecipitation protocol:

  • Membrane solubilization using mild detergents (0.5-1% NP-40, CHAPS, or digitonin)

  • Pre-clearing lysates to reduce non-specific binding

  • Extended incubation periods (overnight at 4°C) to capture low-abundance interactions

• Flow cytometry applications:

  • Detection of surface-exposed epitopes using non-permeabilizing conditions

  • Quantification of expression levels across cell populations

  • Sorting of cells based on expression for downstream analysis

• ELISA-based quantification:

  • Sandwich ELISA using capture and detection antibodies targeting different epitopes

  • Competitive ELISA for measuring binding interactions

  • Time-resolved ELISA for enhanced sensitivity

When working with recombinant tagged versions, researchers can leverage the affinity tag (e.g., His-tag) for detection and purification, complementing antibody-based approaches. Validation of antibody specificity should be performed using knockout or knockdown controls to ensure signal specificity .

How can researchers design experiments to investigate C13orf44/SMIM2 interactions with other proteins?

Investigating protein-protein interactions involving C13orf44/SMIM2 requires specialized approaches that account for its membrane-embedded nature:

• Proximity-based interaction methods:

  • BioID or TurboID proximity labeling to identify proteins in close proximity to C13orf44/SMIM2 in living cells

  • FRET/BRET analysis for real-time interaction monitoring

  • Split-protein complementation assays to verify direct interactions

• Affinity-based methods:

  • Crosslinking mass spectrometry (XL-MS) with membrane-compatible crosslinkers

  • Co-immunoprecipitation with specialized membrane protein extraction buffers

  • Pull-down assays using purified recombinant protein as bait

• Experimental design considerations:

  • Control for tag-mediated artifacts by comparing N- and C-terminally tagged constructs

  • Include appropriate negative controls (e.g., unrelated membrane proteins)

  • Validate interactions through reciprocal pull-downs

• Data analysis and validation:

  • Implement statistical thresholding for mass spectrometry data

  • Validate key interactions through orthogonal methods

  • Assess biological relevance through functional studies

A systematic approach combining multiple complementary techniques provides the most robust characterization of C13orf44/SMIM2's interaction network. This multi-method strategy helps overcome the limitations of individual techniques, particularly for membrane proteins where interactions may be affected by the local lipid environment .

What advanced analytical techniques are recommended for structural studies of C13orf44/SMIM2?

Structural characterization of membrane proteins like C13orf44/SMIM2 presents unique challenges that require specialized approaches:

These techniques should be applied in combination, as each provides complementary information about different aspects of C13orf44/SMIM2 structure and function. Researchers should consider the limitations of each method, particularly regarding sample preparation requirements and resolution limitations for membrane proteins.

What emerging technologies might advance C13orf44/SMIM2 research?

Several cutting-edge technologies show promise for advancing C13orf44/SMIM2 research beyond current methodological limitations:

• AlphaFold2 and structure prediction:

  • Application of AI-based structure prediction to generate working structural models

  • Integration with experimental data for hybrid structural determination

  • Structure-based functional hypothesis generation

• Single-cell multi-omics:

  • Correlation of C13orf44/SMIM2 expression with transcriptomic and proteomic profiles

  • Identification of cell type-specific functions and regulatory networks

  • Characterization of expression heterogeneity in complex tissues

• CRISPR-based functional genomics:

  • High-throughput screening using CRISPR activation/inhibition

  • Base editing for introducing specific mutations to test structure-function hypotheses

  • CRISPRi for temporal control of expression to study dynamic processes

• Advanced imaging techniques:

  • Super-resolution microscopy for nanoscale localization studies

  • Live-cell single-molecule tracking to analyze dynamics

  • Correlative light and electron microscopy for structural contextualization

These emerging approaches, when combined with established methodologies, offer new avenues for understanding C13orf44/SMIM2 function in greater detail and may help resolve outstanding questions in the field .

How can researchers develop evidence-based hypotheses about C13orf44/SMIM2 function?

Developing robust hypotheses about C13orf44/SMIM2 function requires integration of multiple evidence sources through a systematic approach:

• Data integration framework:

  • Compile evidence from structural predictions, expression patterns, and evolutionary conservation

  • Apply weight-of-evidence methodology to evaluate hypothesis strength

  • Identify key knowledge gaps requiring experimental investigation

• Computational prediction approaches:

  • Leverage machine learning algorithms trained on known membrane protein functions

  • Apply network analysis to identify potential functional pathways

  • Use domain-based functional prediction tools

• Experimental validation pipeline:

  • Design experiments with clear discrimination between competing hypotheses

  • Implement hierarchical testing from in vitro to cellular to in vivo systems

  • Establish quantitative metrics for hypothesis evaluation

• Evidence synthesis matrix:

  Evidence Type	Strength	Limitations	Integration Approach
  Sequence homology	Medium	Limited to conserved functions	Weighted with experimental data
  Expression correlation	Medium	Association not causation	Network analysis
  Knockdown phenotypes	High	Potential off-target effects	Validation across models
  Interaction partners	High	May include non-functional interactions	Functional enrichment analysis

This systematic approach to hypothesis development ensures that research directions are grounded in existing evidence while identifying the most promising avenues for new discovery .