Recombinant Kluyveromyces lactis Monopolar spindle protein 2 (MPS2)

How is recombinant K. lactis MPS2 protein typically produced and purified for research applications?

Recombinant K. lactis MPS2 protein is typically produced in E. coli expression systems rather than in its native yeast. The methodology involves cloning the full-length MPS2 gene (1-322 amino acids) into an expression vector with an N-terminal His-tag. After transformation into E. coli and induction of protein expression, the cells are lysed, and the protein is purified through affinity chromatography using the His-tag.

For optimal results, researchers should:

• Perform affinity chromatography using nickel or cobalt resin columns

• Apply additional purification steps like size exclusion chromatography if higher purity is required

• Verify protein identity through mass spectrometry or Western blotting

• Assess purity via SDS-PAGE (should achieve >90% purity)

• Lyophilize the purified protein for long-term storage

The recombinant protein is typically stored as a lyophilized powder and reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol addition for stability during freeze-thaw cycles .

What are the recommended storage and handling conditions for recombinant K. lactis MPS2 protein?

For optimal stability and activity of recombinant K. lactis MPS2 protein, the following storage and handling protocols are recommended:

Before opening the vial, it should be briefly centrifuged to bring contents to the bottom. For experimental use, small working aliquots should be prepared to minimize freeze-thaw cycles. The reconstituted protein should be handled with standard laboratory precautions, including the use of gloves and sterile technique to prevent contamination .

How does MPS2 from K. lactis functionally compare to homologous proteins in other yeast species?

K. lactis MPS2 shares functional homology with spindle pole body (SPB) proteins from other yeast species, but with several distinctive characteristics. While the core function in spindle pole body organization is conserved, K. lactis MPS2 shows evolutionary adaptations specific to this species' unique cell division patterns.

Comparative functional analysis of MPS2 homologs across yeast species:

K. lactis MPS2 is particularly interesting in the context of its adaptation to the specific cellular environment of this species, which has evolved distinct metabolic pathways, including the acquired ability to ferment lactose through evolutionary gene transfer. This suggests that MPS2 may have co-evolved with other cellular systems to accommodate the specific growth and division patterns of K. lactis in its ecological niche .

What experimental approaches are most effective for studying MPS2 protein interactions in K. lactis?

For investigating MPS2 protein interactions in K. lactis, several complementary experimental approaches have proven effective:

• Yeast Two-Hybrid (Y2H) Analysis:

  • Using MPS2 as bait to identify interacting proteins

  • Modified split-ubiquitin Y2H systems recommended for membrane-associated regions

  • Controls must include testing against empty vectors and unrelated proteins

• Co-Immunoprecipitation (Co-IP) with Mass Spectrometry:

  • Using anti-His antibodies to pull down recombinant MPS2 and associated proteins

  • Crosslinking protocols with formaldehyde (1%) can stabilize transient interactions

  • Mass spectrometry analysis of co-precipitated proteins with ≥2 unique peptide matches considered significant

• Fluorescence Microscopy with Tagged Variants:

  • GFP or mCherry fusion constructs for localization studies

  • FRET/FLIM analysis for direct interaction assessment

  • Time-lapse imaging during cell division to track dynamic interactions

• Proximity-Dependent Biotin Identification (BioID):

  • Fusion of biotin ligase (BirA*) to MPS2

  • Allows identification of proximal proteins in native cellular context

  • Particularly useful for detecting weak or transient interactions at the spindle pole body

Each method has specific advantages for detecting different types of interactions. A combination of at least two orthogonal methods is recommended for result validation and comprehensive interaction mapping .

How does the evolutionary history of K. lactis influence the study of MPS2 and what implications does this have for research design?

The evolutionary history of K. lactis, particularly its acquisition of lactose metabolism capabilities, has significant implications for MPS2 research design. K. lactis acquired its ability to ferment lactose through introgression of LAC12 and LAC4 genes from K. marxianus, demonstrating that this yeast has undergone significant genomic changes due to human-mediated selective pressures during domestication.

This evolutionary background necessitates several considerations when designing MPS2 research:

• Strain Selection Considerations:

  • Different K. lactis strains may exhibit variations in MPS2 sequence and regulation

  • Researchers should sequence-verify MPS2 in their specific strain before experimentation

  • Comparative studies with K. marxianus MPS2 may reveal adaptive changes

• Expression System Design:

  • Native promoter studies must account for potential regulatory adaptations

  • Heterologous expression systems should be carefully selected to match codon usage preferences

  • Consider the influence of metabolic state (e.g., lactose vs. glucose growth) on MPS2 expression

• Functional Analysis Framework:

  • Phenotypic assays should include growth on different carbon sources (including lactose)

  • Cell division dynamics may differ between natural isolates and laboratory strains

  • Spindle pole body structure may have co-evolved with metabolic adaptations

• Ecological Context Interpretation:

  • MPS2 function should be interpreted within K. lactis' niche adaptation (dairy environments)

  • Stress response mechanisms may influence MPS2 function differently than in other yeasts

  • Gene expression patterns may reflect adaptation to human-created environments

This evolutionary perspective suggests that MPS2 studies should incorporate comparative approaches and consider the unique genomic history of K. lactis, especially when making functional inferences across species .

What are the optimal conditions for expressing recombinant K. lactis MPS2 to maximize yield and solubility?

Optimizing expression of recombinant K. lactis MPS2 requires careful consideration of several parameters to balance yield with proper folding and solubility. Based on experimental data, the following conditions have been shown to be most effective:

The membrane-associated C-terminal domain of MPS2 can contribute to aggregation and inclusion body formation. To address this challenge:

• Consider expressing the N-terminal domain (residues 1-200) separately if the full-length protein shows poor solubility

• Add 0.1% mild detergents (Triton X-100 or n-dodecyl β-D-maltoside) to lysis buffers

• Include 10% glycerol in all buffers to stabilize the protein

• Use a step-wise purification protocol with increasing imidazole concentrations (10mM, 30mM, 250mM)

• Consider on-column refolding protocols if inclusion bodies form despite optimization

These conditions typically yield 5-8 mg of purified protein per liter of culture with >90% purity as assessed by SDS-PAGE .

What experimental approaches can differentiate the specific functions of MPS2 in K. lactis compared to its homologs in other yeast species?

Differentiating the specific functions of MPS2 in K. lactis from its homologs in other yeast species requires multi-faceted experimental approaches:

• Complementation Assays:

  • Express K. lactis MPS2 in MPS2-deficient S. cerevisiae strains

  • Assess the degree of functional rescue under various growth conditions

  • Construct chimeric proteins with domain swapping between homologs to identify species-specific functional domains

  • Quantitative growth curve analysis with measurements taken every 30 minutes for 48 hours

• High-Resolution Microscopy:

  • Super-resolution imaging (PALM/STORM, SIM, or STED) of fluorescently tagged MPS2

  • 3D reconstruction of spindle pole body structure in both species

  • Co-localization studies with other SPB proteins to identify differential interaction patterns

  • Live-cell imaging during cell division with ≤5-minute intervals

• Proteomic Approaches:

  • Comparative interactome mapping using BioID or proximity labeling

  • Quantitative analysis of post-translational modifications by mass spectrometry

  • Hydrogen-deuterium exchange mass spectrometry to compare structural dynamics

  • Cross-species pull-down assays to identify differential binding partners

• Functional Genomics:

  • CRISPR-Cas9 genome editing to create specific domain mutations

  • Synthetic genetic array analysis to map genetic interaction networks

  • Transcriptome analysis under various stress conditions to identify differential regulatory networks

  • Phenotypic profiling using high-content screening with automated image analysis

• Biochemical Characterization:

  • In vitro reconstitution assays with purified components

  • Membrane interaction studies using liposome binding assays

  • Microtubule nucleation and dynamics assays in cell-free systems

  • Comparative structural analysis using cryo-EM or X-ray crystallography if feasible

These approaches should be applied in parallel, with results integrated through bioinformatic analysis to develop a comprehensive model of K. lactis MPS2 function that distinguishes it from homologs in other species .

What are the key considerations for designing site-directed mutagenesis experiments to probe functional domains of K. lactis MPS2?

Designing effective site-directed mutagenesis experiments for K. lactis MPS2 requires careful consideration of structural features, evolutionary conservation, and experimental validation approaches. Here are the key considerations:

• Target Selection Strategy:

  Domain Type	Residue Selection Criteria	Mutation Type
  Conserved motifs	>90% identity across yeasts	Alanine scanning
  Species-specific regions	Unique to K. lactis	Domain swapping
  Predicted functional sites	Based on structure prediction	Conservative vs. non-conservative
  Post-translational modification sites	Mass spec identified	Phospho-mimetic (S/T→D/E)
  Membrane interaction domains	Hydrophobicity analysis	Charge inversion

• Critical Residues for Initial Focus:

  • Positions 45-62: Potential protein-protein interaction domain

  • Positions 180-195: Highly conserved region likely involved in core function

  • Positions 275-290: Membrane-spanning domain with species-specific features

  • Specific residues S57, T124, and Y250: Potential regulatory phosphorylation sites

• Validation Approaches:

  • Functional complementation assays in MPS2-deleted strains

  • Fluorescence microscopy to assess localization

  • Co-immunoprecipitation to test interaction with known partners

  • Growth phenotype analysis under various stress conditions

  • Cell cycle progression analysis with flow cytometry

• Controls and Experimental Design:

  • Include wild-type MPS2 and empty vector controls

  • Create both single mutations and combined mutations

  • Use quantitative rather than qualitative assessments

  • Perform rescue experiments with wild-type when possible

  • Test function under multiple conditions (temperature, media, cell cycle phases)

• Advanced Approaches:

  • Consider sequential multi-site mutagenesis for complex functional mapping

  • Develop inducible or temperature-sensitive mutants for temporal studies

  • Combine mutagenesis with high-throughput screening methods

  • Use deep mutational scanning for comprehensive functional mapping

  • Apply structural modeling to predict and interpret mutational effects

When designing primers for mutagenesis, ensure at least 15-20 bp of homology on either side of the mutation site and verify the absence of unwanted secondary structures. After mutagenesis, sequence the entire MPS2 gene to confirm the presence of only the intended mutations .

How can researchers distinguish between direct and indirect effects when studying MPS2 function in K. lactis cellular processes?

Distinguishing between direct and indirect effects of MPS2 on K. lactis cellular processes requires a multi-layered experimental approach that combines immediate biochemical interactions with broader cellular phenotypes:

• Temporal Resolution Approaches:

  • Develop an auxin-inducible degron (AID) system for MPS2 to achieve rapid protein depletion

  • Perform time-course experiments with sampling at short intervals (5, 15, 30, 60, 120 minutes)

  • Use real-time imaging with fluorescent reporters to track immediate consequences

  • Compare rapid vs. gradual depletion phenotypes to separate primary from secondary effects

• Spatial Resolution Methods:

  • Employ proximity labeling (BioID or APEX) to identify proteins within a 10-20 nm radius of MPS2

  • Use subcellular fractionation to track changes in protein distribution

  • Apply correlative light and electron microscopy (CLEM) to link ultrastructural changes to MPS2 localization

  • Perform spatially restricted optogenetic control of MPS2 function

• Biochemical Interaction Authentication:

  • Validate direct interactions with purified components in vitro

  • Use microscale thermophoresis or surface plasmon resonance to measure binding kinetics

  • Perform crosslinking mass spectrometry to map interaction interfaces

  • Employ hydrogen-deuterium exchange mass spectrometry to identify conformational changes

• Network-Based Discrimination:

  • Map genetic interaction profiles and compare to physical interaction networks

  • Identify synthetic lethal and synthetic rescue interactions

  • Perform epistasis analysis with known spindle pole body components

  • Use Bayesian network modeling to infer causal relationships

• Analytical Framework for Data Integration:

  Data Type	Direct Effect Indicators	Indirect Effect Indicators
  Temporal	Occurs within minutes	Requires hours to manifest
  Proximity	Physical contact or <10nm	Separated by >20nm
  Dependency	Persists in diverse genetic backgrounds	Varies with genetic context
  Biochemical	Reconstitutable with purified components	Requires cellular context
  Specificity	Affected by point mutations	Requires domain deletion

By integrating these approaches, researchers can build a hierarchical model of MPS2-dependent processes, distinguishing between primary interactions and downstream consequences .

What are the challenges in interpreting MPS2 localization data, and how can researchers overcome these limitations?

Interpreting MPS2 localization data presents several technical and biological challenges that require specific methodological solutions:

• Resolution Limitations in Conventional Microscopy:

  • Challenge: The spindle pole body is typically 80-120 nm in diameter, below the resolution limit of conventional light microscopy

  • Solution: Implement super-resolution techniques such as PALM/STORM (≤20 nm resolution) or structured illumination microscopy (SIM, ≤100 nm resolution)

  • Validation: Correlative light and electron microscopy to precisely position MPS2 within the ultrastructure

• Signal-to-Noise Ratio Problems:

  • Challenge: MPS2 is not abundant, with typically 100-150 molecules per cell

  • Solution: Use brighter fluorophores (e.g., mNeonGreen) or signal amplification methods

  • Quantification: Apply deconvolution algorithms and measure signal-to-noise ratios (aim for >5:1)

  • Control: Include measurements of cellular autofluorescence baseline

• Tag-Induced Artifacts:

  • Challenge: Fluorescent protein tags (25-30 kDa) may interfere with MPS2 function

  • Solution: Compare N- and C-terminal tagging, use smaller tags (e.g., SNAP or HaloTag)

  • Validation: Perform complementation assays to verify functionality of tagged variants

  • Alternative: Use antibody staining of endogenous protein when possible

• Dynamic Localization Challenges:

  • Challenge: MPS2 may relocalize during cell cycle phases

  • Solution: Synchronize cultures or use cell cycle stage markers

  • Approach: Implement 4D imaging (x, y, z, time) with ≤3-minute intervals

  • Analysis: Develop automated tracking algorithms to follow spindle pole body movement

• Reproducibility and Quantification Issues:

  • Challenge: Variability between experiments and subjective interpretation

  • Solution: Standardize image acquisition parameters and processing workflows

  • Requirement: Analyze ≥100 cells per condition across ≥3 biological replicates

  • Approach: Implement automated, unbiased quantification methods with defined thresholds

• Interpretation Framework:

  Observation	Potential Interpretation	Verification Approach
  Diffuse cytoplasmic signal	Misfolding or overexpression	Titrate expression levels
  Multiple foci	Aggregation or multiple functions	Co-localize with other markers
  Cell cycle-dependent patterns	Regulated function	Synchronize with multiple methods
  Strain-dependent variation	Genetic background effects	Test in defined genetic contexts

By addressing these challenges systematically, researchers can ensure that MPS2 localization data accurately reflects the protein's true biological distribution and function, rather than technical artifacts .

How can researchers integrate data from different experimental approaches to build a comprehensive model of MPS2 function in K. lactis?

Building a comprehensive model of MPS2 function in K. lactis requires systematic integration of diverse experimental datasets. Here is a structured approach to data integration:

• Hierarchical Data Organization:

  • Organize data into tiers based on evidence strength (direct biochemical > genetic interaction > correlative)

  • Classify observations as structural, functional, or regulatory

  • Create interaction maps with confidence scores for each interaction

  • Develop a central database to store all experimental results with standardized metadata

• Multi-scale Integration Framework:

  Scale	Data Types	Integration Methods
  Molecular	Structural, biochemical, biophysical	Molecular modeling and docking
  Subcellular	Localization, proximity, dynamics	Spatial network mapping
  Cellular	Phenotypic, transcriptomic, proteomic	Network analysis, pathway mapping
  Population	Growth rates, stress responses	Fitness landscape modeling

• Computational Integration Approaches:

  • Apply Bayesian network modeling to infer causal relationships

  • Use machine learning to identify patterns across datasets

  • Develop predictive models testable with new experiments

  • Perform sensitivity analysis to identify critical parameters

  • Implement graph theory approaches to map interaction networks

• Validation Strategy:

  • Design experiments specifically to test integrated model predictions

  • Identify and resolve contradictions between different data types

  • Use orthogonal methods to confirm key model components

  • Develop quantitative metrics to assess model performance

• Visualization and Communication Tools:

  • Create interactive visualizations of the integrated model

  • Develop standardized nomenclature for model components

  • Generate both simplified models for conceptual understanding and detailed models for computational analysis

  • Document model assumptions and limitations explicitly

• Iterative Refinement Process:

  • Establish a systematic workflow for model updating as new data becomes available

  • Prioritize experiments based on model uncertainties

  • Track model versions and performance metrics over time

  • Develop quantitative criteria for model improvement

This integration approach should be applied iteratively, with the model being refined as new data becomes available. The goal is to develop a mechanistic understanding of MPS2 function that explains existing observations and can predict outcomes of new perturbations. The most robust elements of the model will be those supported by multiple independent experimental approaches .

What emerging technologies could advance our understanding of K. lactis MPS2 function beyond current methodological limitations?

Several cutting-edge technologies show promise for overcoming current limitations in studying K. lactis MPS2:

• Cryo-Electron Tomography (Cryo-ET):

  • Enables visualization of MPS2 in its native cellular context at near-atomic resolution

  • Can reveal detailed structural organization within the spindle pole body

  • Requires technical advances in sample preparation for yeast cells

  • Expected resolution improvement: From current ~2-3 nm to sub-nanometer precision

• Live-Cell Single-Molecule Tracking:

  • Allows tracking of individual MPS2 molecules in living cells

  • Can reveal dynamic behaviors and transient interactions

  • Requires development of photoconvertible fluorescent tags compatible with K. lactis

  • Target temporal resolution: 10-20 milliseconds per frame

• Genome-Wide CRISPR Screening in K. lactis:

  • Systematic identification of genetic interactions with MPS2

  • Requires optimization of CRISPR-Cas9 systems for efficient editing in K. lactis

  • Development of K. lactis-specific sgRNA libraries

  • Goal: >90% genome coverage with <5% false discovery rate

• Spatial Transcriptomics and Proteomics:

  • Maps transcriptome/proteome changes with subcellular resolution

  • Can reveal localized responses to MPS2 perturbation

  • Requires adaptation of protocols for yeast cell architecture

  • Spatial resolution target: <500 nm regions within single cells

• Protein Structure Prediction with AI:

  • Advanced protein structure prediction tools (e.g., AlphaFold2) applied to MPS2

  • Can generate structural models to guide experimental design

  • Integration with molecular dynamics simulations

  • Accuracy goal: <2Å RMSD for structural predictions

• Cell-Free Reconstitution Systems:

  • In vitro reconstitution of minimal functional units containing MPS2

  • Bottom-up approach to identify essential components

  • Allows manipulation of system composition with precise control

  • Challenge: Maintaining membrane-associated functions in reconstituted systems

• Optogenetic and Chemical-Genetic Tools:

  • Development of K. lactis-optimized optogenetic controllers for MPS2

  • Creation of rapid, reversible perturbation systems

  • Enables precise temporal control of MPS2 function or interactions

  • Target activation/inactivation time: <1 minute

These emerging technologies, particularly when used in combination, have the potential to provide unprecedented insights into MPS2 function, addressing current limitations in spatial resolution, temporal dynamics, and system complexity .

How might comparative studies of MPS2 across Kluyveromyces species contribute to our understanding of spindle pole body evolution?

Comparative studies of MPS2 across Kluyveromyces species offer a unique opportunity to understand spindle pole body (SPB) evolution in the context of yeast adaptation to diverse ecological niches:

• Evolutionary Rate Analysis:

  • Compare substitution rates in MPS2 across Kluyveromyces species

  • Identify regions under purifying selection (functionally constrained) versus positive selection (adaptation)

  • Use dN/dS ratios to quantify selection pressure on different domains

  • Correlate evolutionary rates with structural features and known functions

• Functional Diversification Mapping:

  • Compare MPS2 functions in species with different ecological adaptations:

    • K. lactis: Dairy environments, lactose utilization

    • K. marxianus: Diverse substrates, thermotolerance

    • K. dobzhanskii: Plant surfaces, stress resistance

    • K. aestuarii: Marine environments, osmotolerance

  • Test cross-species complementation to identify species-specific functions

  • Correlate functional differences with ecological adaptations

• Structural Comparison Framework:

  Species	Predicted Structural Features	Correlation with Ecological Niche
  K. lactis	Standard SPB architecture, moderate size	Stable dairy environment adaptation
  K. marxianus	Potentially reinforced structure	Adaptation to temperature fluctuations
  K. dobzhanskii	Possibly modified outer plaque	Plant surface attachment specialization
  K. aestuarii	Predicted membrane adaptations	Osmotic stress resistance

• Genomic Context Analysis:

  • Compare synteny around MPS2 loci across species

  • Identify co-evolved gene clusters related to cell division

  • Map chromosomal rearrangements that may have affected MPS2 regulation

  • Analyze promoter evolution to identify regulatory changes

• Interactome Evolution:

  • Map MPS2 interaction partners across species

  • Identify conserved versus species-specific interactions

  • Correlate interactome changes with SPB structural adaptations

  • Reconstruct the ancestral SPB interactome

• Integration with Phylogenetic Analysis:

  • Reconstruct MPS2 evolutionary history within fungal lineage

  • Correlate major evolutionary transitions with ecological shifts

  • Apply molecular clock approaches to date key adaptations

  • Compare with evolution of other SPB components to identify co-evolution patterns

This comparative approach can reveal how selective pressures in different environments have shaped MPS2 evolution, providing insights into the fundamental mechanisms of spindle pole body function and adaptation. Understanding these evolutionary patterns may also help explain why certain features are conserved across vast evolutionary distances while others have undergone rapid diversification .

What are the potential applications of understanding K. lactis MPS2 for developing novel research tools or biotechnological applications?

Understanding K. lactis MPS2 could lead to several innovative research tools and biotechnological applications:

• Engineered Cell Division Control Systems:

  • Development of inducible MPS2 variants to synchronize yeast cultures

  • Creation of temperature-sensitive MPS2 mutants for precise cell cycle control

  • Applications in standardized yeast biomass production and fermentation processes

  • Potential for regulating cell division timing in biotechnological processes

• Novel Protein Expression Platforms:

  • Exploitation of MPS2-based anchoring systems for protein immobilization

  • Development of SPB-targeted protein production for difficult-to-express proteins

  • Creation of spatial segregation systems for compartmentalized biochemical reactions

  • Improvement of K. lactis as a protein expression host for biotechnology

• Biosensors and Diagnostic Tools:

  • MPS2-based biosensors for monitoring cell cycle progression

  • Development of yeast-based screening systems for anti-mitotic compounds

  • Creation of reporters for environmental stress detection

  • Potential applications in pharmaceutical screening and environmental monitoring

• Synthetic Biology Applications:

  • Incorporation of MPS2 domains into synthetic protein scaffolds

  • Engineering of artificial organelle-like structures in yeast

  • Development of programmable cellular architecture systems

  • Applications in creating multi-enzyme complexes for biocatalysis

• Evolutionary and Comparative Genomics Tools:

  • MPS2 as a marker for evolutionary studies in fungi

  • Development of species-specific detection methods based on MPS2 sequence

  • Creation of taxonomic classification tools for Kluyveromyces species

  • Applications in food microbiology and quality control

• Specialized Research Reagents:

  Reagent Type	Potential Applications	Development Stage
  Anti-MPS2 antibodies	Immunoprecipitation, microscopy	Feasible with purified protein
  Fluorescent MPS2 probes	Live-cell imaging, SPB tracking	Requires optimization for K. lactis
  MPS2 domain peptides	Inhibition studies, interaction mapping	Requires domain function characterization
  MPS2 expression vectors	Heterologous expression, structure studies	Immediately developable

• Bioprocessing Improvements:

  • Engineering of MPS2 to enhance stress resistance in industrial K. lactis strains

  • Development of growth optimization strategies for biotechnological applications

  • Improvement of culture synchronization for standardized product output

  • Potential applications in cheese and kefir production processes

Understanding the fundamental biology of MPS2 in K. lactis could thus lead to diverse applications ranging from basic research tools to industrial bioprocessing improvements. The unique properties of K. lactis as a generally recognized as safe (GRAS) organism make it particularly suitable for food-related and pharmaceutical applications derived from this research .