Recombinant Macaca fascicularis Metaxin-1 (MTX1)

What is Metaxin-1 and what is its primary function in cellular systems?

Metaxin-1 (MTX1) is a protein encoded by the MTX1 gene that plays a critical role in mitochondrial protein import. It functions as a component of the mitochondrial outer membrane import complex, facilitating the translocation of proteins from the cytosol into mitochondria . MTX1 is particularly involved in the metabolism of proteins and mitochondrial protein import pathways . The protein interacts with several other proteins including TMBIM4, MTX2, 1C, IMMT, and SAMM50 , forming important complexes that maintain mitochondrial integrity and function. Understanding the structure-function relationship of MTX1 requires consideration of its membrane topology and interaction domains that enable its participation in protein transport machinery.

How does Macaca fascicularis MTX1 compare structurally to human MTX1?

Macaca fascicularis (cynomolgus macaque) MTX1 shares high sequence homology with human MTX1, making it an excellent model for biomedical research . The evolutionary conservation of MTX1 across primates reflects its essential biological function. Researchers can utilize high-quality genome assemblies, such as the chromosome-level assembly for cynomolgus macaque developed using PacBio, 10x Genomics, HiC, and HiSeq technologies , to conduct comparative sequence analyses. This assembly spans 5.1 Gb with 16,741 contigs and provides higher confidence in the genetic architecture of the cynomolgus macaque genome . Detailed structural comparison reveals conserved functional domains between human and macaque MTX1, although species-specific variations may impact certain protein-protein interactions or regulatory mechanisms.

What expression systems are most effective for producing recombinant Macaca fascicularis MTX1?

For recombinant expression of Macaca fascicularis MTX1, researchers have successfully employed several systems including:

The choice of expression system depends on the experimental requirements. For structural studies requiring high yields, E. coli systems with appropriate solubility tags (His, T7) may be preferred . For functional studies where post-translational modifications are critical, mammalian expression systems provide more physiologically relevant protein. Codon optimization for the respective expression system significantly improves yield, while fusion tags facilitate purification and can enhance solubility.

What purification strategies optimize yield and activity of recombinant MTX1?

Purification of recombinant MTX1 requires a multi-step approach:

• Initial Capture: Affinity chromatography using His-tag, T7, or other fusion tags facilitates selective binding of the target protein .

• Intermediate Purification: Ion exchange chromatography separates proteins based on charge differences.

• Polishing: Size exclusion chromatography removes aggregates and provides buffer exchange.

Maintaining protein stability throughout purification is critical. Addition of protease inhibitors, working at 4°C, and optimizing buffer conditions (pH, salt concentration, reducing agents) preserves structural integrity and activity. For membrane-associated proteins like MTX1, inclusion of mild detergents or amphipols during purification helps maintain native conformation. Validation of protein quality through analytical techniques (SDS-PAGE, Western blotting, mass spectrometry) ensures consistency between preparations.

How can recombinant MTX1 be utilized in studying mitochondrial protein import mechanisms?

Recombinant MTX1 serves as a valuable tool for investigating mitochondrial protein import mechanisms through several experimental approaches:

• In vitro reconstitution assays: Purified recombinant MTX1 can be incorporated into liposomes or nanodiscs to reconstruct the protein import machinery. This system allows researchers to study the protein's role in translocation events under controlled conditions.

• Interaction mapping: Using recombinant MTX1 as bait in pull-down assays or co-immunoprecipitation experiments helps identify interaction partners in the mitochondrial import pathway. The known interactions with TMBIM4, MTX2, 1C, IMMT, and SAMM50 provide a foundation for building comprehensive interaction networks.

• Structure-function analysis: Site-directed mutagenesis of recombinant MTX1 followed by functional assays reveals critical residues involved in protein-protein interactions or substrate recognition.

• Comparative studies: Parallel analysis of human and Macaca fascicularis MTX1 highlights conserved mechanisms and species-specific adaptations in mitochondrial protein import.

These approaches collectively contribute to understanding the molecular mechanisms of mitochondrial protein import and the specific role of MTX1 in this essential cellular process.

What experimental controls are essential when working with recombinant Macaca fascicularis MTX1?

Robust experimental design requires appropriate controls to ensure validity and reproducibility:

• Negative controls:

  • Empty vector-transfected cells to account for expression system artifacts

  • Heat-denatured recombinant MTX1 to distinguish specific from non-specific effects

  • Non-related proteins of similar size/structure to validate binding specificity

• Positive controls:

  • Human MTX1 for comparative analysis

  • Known interaction partners like TMBIM4 or MTX2 to validate assay functionality

  • Well-characterized mitochondrial import substrates

• Validation controls:

  • Multiple detection methods (e.g., different antibodies, tags)

  • Reciprocal interaction assays (pull-down from both directions)

  • Dose-response relationships to confirm specificity

• Technical controls:

  • Protein quality assessment (purity, stability, activity)

  • Batch-to-batch consistency verification

  • Environmental condition standardization (temperature, pH, ionic strength)

Implementation of these controls ensures that experimental findings are attributable to the biological properties of MTX1 rather than technical artifacts or experimental design flaws.

How does post-translational modification affect MTX1 function in mitochondrial protein import?

Post-translational modifications (PTMs) of MTX1 represent a sophisticated regulatory layer controlling mitochondrial protein import. Research methodologies to investigate PTMs include:

• Identification of PTM sites: Mass spectrometry-based proteomics can map phosphorylation, acetylation, ubiquitination, and other modifications on recombinant or endogenous MTX1.

• Functional impact assessment: Site-directed mutagenesis of modified residues (mimicking or preventing modifications) followed by functional assays reveals how PTMs affect:

  • Protein-protein interactions with import machinery components

  • Substrate recognition specificity

  • Conformational dynamics during import cycles

• Regulation under stress conditions: Exposing cells to oxidative stress, nutrient deprivation, or other stressors can trigger dynamic changes in MTX1 modification patterns, potentially as adaptive responses to maintain mitochondrial homeostasis.

• Enzyme identification: Biochemical approaches combined with genetic screens help identify the kinases, phosphatases, acetyltransferases, or other enzymes responsible for MTX1 modification and demodification.

Understanding the PTM landscape of MTX1 provides insights into how cells dynamically regulate mitochondrial protein import in response to changing physiological conditions or stress.

What approaches can resolve contradictory data regarding MTX1 interactions in different experimental systems?

Resolving contradictory experimental data requires systematic troubleshooting and methodological refinement:

• System-specific variables: Different expression systems (mammalian, bacterial, insect cells) may influence protein folding, modifications, and interaction capabilities of MTX1. Parallel testing in multiple systems with standardized conditions helps identify system-dependent artifacts.

• Methodological triangulation: Employing complementary techniques to study the same interaction:

  • In vitro: Surface plasmon resonance, isothermal titration calorimetry

  • Cell-based: FRET, BiFC, co-immunoprecipitation

  • In silico: Molecular docking, molecular dynamics simulations

• Proximity-based mapping: Techniques like BioID or APEX proximity labeling provide spatial context for interactions, distinguishing direct from indirect associations within the mitochondrial import complex.

• Stoichiometry and kinetics analysis: Quantitative binding assays with varying concentrations and time courses reveal whether contradictory results stem from differences in binding affinities or association/dissociation kinetics.

• Structural biology approaches: Cryo-EM or X-ray crystallography of MTX1 complexes provides definitive evidence for interaction interfaces and conformational states.

Systematic documentation of experimental conditions, reagent sources, and analytical parameters facilitates troubleshooting and reconciliation of seemingly contradictory results.

How does the genomic context of MTX1 in Macaca fascicularis inform its functional conservation?

The genomic context of MTX1 in Macaca fascicularis provides valuable evolutionary insights:

The recently developed high-quality, phased hybrid genomic assembly for cynomolgus macaque with chromosome-length scaffolds offers unprecedented resolution for genomic analysis . This assembly correctly identifies structural variations not present in previous assemblies and provides a foundation for comparative genomic studies .

Researchers can examine:

• Syntenic relationships: Conservation of gene order and neighboring genes around MTX1 between macaque and human genomes suggests preserved regulatory contexts.

• Regulatory element conservation: Analysis of promoter regions, enhancers, and transcription factor binding sites reveals evolutionary pressure on expression regulation.

• Alternative splicing patterns: RNA-seq data from different tissues can identify species-specific splicing events that might generate functionally distinct MTX1 isoforms.

• Selection pressure analysis: Calculation of dN/dS ratios (nonsynonymous to synonymous substitution rates) across primate MTX1 orthologs highlights domains under purifying or positive selection.

The cynomolgus macaque assembly identified novel inversions with high spatial resolution, providing confidence in the structural variability identified . This genomic context analysis contributes to understanding both functional conservation and species-specific adaptations of MTX1.

What are the key differences in experimental approaches when studying recombinant versus endogenous MTX1?

Studying recombinant versus endogenous MTX1 involves distinct methodological considerations:

When transitioning between systems:

• Validation strategies: Confirming that observations with recombinant protein reflect endogenous behavior through parallel experiments.

• Complementation approaches: Testing whether recombinant MTX1 restores function in cells with depleted endogenous protein.

• Quantitative considerations: Accounting for potential artifacts from non-physiological expression levels of recombinant protein.

• Species-specific differences: When using macaque MTX1 in human cell systems, considering potential cross-species compatibility issues.

Understanding these differences enables researchers to design experiments that leverage the advantages of each approach while mitigating their limitations.

What are common challenges in expressing soluble, functional Macaca fascicularis MTX1 and their solutions?

Researchers frequently encounter specific challenges when expressing recombinant MTX1:

• Insolubility and aggregation:

  • Challenge: As a mitochondrial membrane-associated protein, MTX1 may form insoluble aggregates when overexpressed.

  • Solutions: Reduce expression temperature (16-20°C), use solubility-enhancing fusion partners (SUMO, MBP), incorporate mild detergents or amphipols, optimize buffer conditions (pH, salt concentration, reducing agents).

• Improper folding:

  • Challenge: Expression in prokaryotic systems may lead to misfolding due to lack of eukaryotic chaperones.

  • Solutions: Co-express with molecular chaperones, use eukaryotic expression systems, implement slow induction protocols, add chemical chaperones to media.

• Proteolytic degradation:

  • Challenge: Recombinant MTX1 may be susceptible to proteolysis during expression or purification.

  • Solutions: Add protease inhibitors, use protease-deficient host strains, optimize harvest timing, design constructs that exclude protease-sensitive regions.

• Low yield:

  • Challenge: Membrane-associated proteins often express at lower levels than soluble proteins.

  • Solutions: Codon optimization, use strong inducible promoters, scale up culture volume, optimize cell lysis conditions, develop efficient purification protocols.

Systematic optimization using design of experiments (DoE) approaches enables efficient identification of optimal expression conditions specific to Macaca fascicularis MTX1.

How can researchers validate the functional integrity of purified recombinant MTX1?

Validating functional integrity requires multi-parameter assessment:

• Structural integrity:

  • Circular dichroism spectroscopy to verify secondary structure content

  • Thermal shift assays to assess protein stability

  • Dynamic light scattering to evaluate homogeneity and detect aggregation

• Biochemical functionality:

  • ATP hydrolysis assays if applicable

  • Binding assays with known interaction partners (TMBIM4, MTX2, IMMT, SAMM50)

  • Limited proteolysis to confirm proper folding (properly folded proteins show distinctive digestion patterns)

• Cellular functionality:

  • Complementation assays in MTX1-depleted cells

  • Mitochondrial protein import assays using in vitro systems

  • Subcellular localization studies to confirm proper targeting

• Comparative benchmarking:

  • Side-by-side testing with human MTX1 or other well-characterized orthologs

  • Comparison to endogenous protein activity where possible

Establishing clear acceptance criteria for each validation parameter ensures consistent quality across different preparations and experimental settings.

How should researchers interpret MTX1 functional data in the context of mitochondrial disease models?

Interpretation of MTX1 functional data in mitochondrial disease contexts requires careful consideration:

• Pathway integration: MTX1 functions within interconnected mitochondrial processes including protein import and metabolism . Experimental findings should be interpreted within this network perspective, considering compensatory mechanisms and pathway redundancies.

• Species-specific differences: While Macaca fascicularis serves as an excellent model for human biology , subtle differences in mitochondrial biology might influence the translational relevance of findings. The high-quality cynomolgus macaque genome assembly provides a foundation for identifying species-specific variations .

• Quantitative considerations: Establish dose-response relationships and determine threshold effects. MTX1 dysfunction might need to reach critical levels before manifesting phenotypic consequences due to biological buffering capacity.

• Temporal dynamics: Acute versus chronic MTX1 dysfunction may trigger different cellular responses. Time-course experiments help distinguish primary from secondary effects.

• Tissue-specific contexts: The significance of MTX1 alterations may vary across tissues depending on mitochondrial content, metabolic demands, and compensatory capacity.

When linking experimental findings to disease mechanisms, researchers should differentiate between correlation and causation, ideally validating key findings across multiple model systems and methodological approaches.

What statistical approaches are most appropriate for analyzing protein-protein interaction data involving MTX1?

Protein-protein interaction (PPI) data analysis requires specialized statistical approaches:

• Significance testing for co-immunoprecipitation/pull-down experiments:

  • Student's t-test or ANOVA for comparing interaction intensities across conditions

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) when normality assumptions are violated

  • Multiple testing correction (Bonferroni, Benjamini-Hochberg) when screening numerous potential interactors

• Network analysis for interactome studies:

  • Calculation of centrality measures (degree, betweenness, closeness) to assess MTX1's position in interaction networks

  • Cluster detection algorithms to identify functional modules

  • Permutation tests to evaluate network property significance

• Quantitative binding parameters:

  • Nonlinear regression for determining binding affinities (Kd)

  • Scatchard or Hill plot analysis for detecting cooperative binding

  • Global fitting approaches for complex binding models

• False discovery rate control:

  • Implementation of appropriate controls (IgG pull-downs, scrambled peptides)

  • Calculation of SAINT scores or similar metrics to distinguish specific from non-specific interactions

  • Establishment of significance thresholds based on empirical background distributions

• Visualization approaches:

  • Heat maps for displaying interaction intensities across conditions

  • Volcano plots for highlighting significant changes in interaction profiles

  • Network diagrams that incorporate interaction confidence metrics

What documentation should researchers maintain when working with recombinant proteins for NIH-funded projects?

Comprehensive documentation is essential for research reproducibility and regulatory compliance:

• Experimental protocols: Detailed methods for expression, purification, and functional characterization of recombinant Macaca fascicularis MTX1, including:

  • Expression vector construction

  • Host strain/cell line details

  • Induction conditions

  • Purification parameters

  • Quality control metrics

• NIH training grant requirements: Documentation that may be relevant for NIH-funded projects involving recombinant proteins includes:

  • Table 5 for training grants: Publications of trainees supported by the program

  • Table 8 for training grants: Program outcomes demonstrating research productivity

• Biosafety documentation:

  • Risk assessment for recombinant protein work

  • Institutional Biosafety Committee approvals where applicable

  • Standard Operating Procedures (SOPs) for safe handling

• Quality control records:

  • Purity assessments (SDS-PAGE, HPLC)

  • Identity confirmation (mass spectrometry, Western blotting)

  • Functional validation assays

  • Batch records tracking production parameters

• Data management:

  • Raw data storage and backup procedures

  • Analysis workflows with version control

  • Material sharing documentation

Properly maintained documentation not only facilitates regulatory compliance but also enhances research reproducibility and enables effective knowledge transfer within research teams.

What training is recommended for researchers beginning work with recombinant mitochondrial proteins like MTX1?

Comprehensive training for researchers working with recombinant mitochondrial proteins should include:

• Technical skills development:

  • Molecular cloning techniques for expression vector construction

  • Protein expression systems (prokaryotic and eukaryotic)

  • Purification methods (affinity chromatography, size exclusion, ion exchange)

  • Analytical techniques (SDS-PAGE, Western blotting, mass spectrometry)

  • Mitochondrial isolation and subfractionation

• Theoretical knowledge:

  • Mitochondrial biology fundamentals

  • Protein import pathways and mechanisms

  • Structural biology of membrane proteins

  • Comparative primate genomics for those working with Macaca fascicularis proteins

• Safety training:

  • Biosafety principles for recombinant protein work

  • Chemical safety for purification reagents

  • Laboratory-specific standard operating procedures

• Regulatory awareness:

  • NIH guidelines for research involving recombinant molecules

  • Institutional requirements for training grant documentation

  • Data sharing and material transfer policies

• Data analysis competencies:

  • Statistical methods for protein interaction studies

  • Bioinformatics tools for sequence analysis and structural prediction

  • Data visualization techniques

Training should be documented according to institutional requirements, which may include formal courses, workshops, or mentored laboratory experiences. For NIH-funded projects, this training documentation may become part of the reporting requirements outlined in the Data Tables framework .