Recombinant UPF0057 membrane protein ZK632.10 (ZK632.10)

What is UPF0057 membrane protein ZK632.10 and what organism does it originate from?

UPF0057 membrane protein ZK632.10 is a small hydrophobic membrane protein belonging to the Plasma Membrane Proteolipid 3 (PMP3) family. It originates from the nematode Caenorhabditis elegans and consists of 80 amino acids with a UniProt ID of P34655. The protein contains two predicted transmembrane helices, which is characteristic of the PMP3/UPF0057 family. The full amino acid sequence is: MCQILLAILAIFLPPIAVLLDVGCNCDLLINILLTCLGIIPGIIHAWYIILCKEKTVVQNIYVQTNDHGTAPPAYSPYSA .

How does ZK632.10 compare to other members of the UPF0057 family?

ZK632.10 belongs to Group I PMP3 homologues (PMP3(i)hs), which are expressed abundantly during normal vegetative growth in both prokaryotic and eukaryotic cells. Like other members of this family, it features a compact structure with two transmembrane domains. Comparative studies have shown that despite sequence variations across different species, the functional properties of PMP3/UPF0057 proteins are remarkably conserved, with eukaryotic homologues capable of functionally suppressing conditional growth defects in bacterial deletion mutants, demonstrating conserved cross-kingdom membrane functions .

What are the predicted structural features of ZK632.10?

Based on sequence analysis and comparative studies with other PMP3 family members, ZK632.10 is predicted to have:

• Two transmembrane helices

• A total length of 80 amino acids

• Predominantly hydrophobic residues, particularly in the transmembrane regions

• Potential lipid-binding selectivity

• Membrane localization determinants

These structural features contribute to its membrane integration and functionality in regulating membrane potential and ionic homeostasis .

What expression systems are most effective for producing recombinant ZK632.10?

Multiple expression systems have been evaluated for UPF0057 family membrane proteins, with varying advantages:

E. coli System:

• Highest yield (5-50 mg per liter of culture)

• Often directs proteins to inclusion bodies, requiring refolding

• Most economical and rapid production system

• Suitable when post-translational modifications aren't critical

Insect Cell/Baculovirus System:

• Provides eukaryotic post-translational modifications

• Better folding for complex membrane proteins

• More native-like membrane environment

• Optimized using the Vertiga-IM platform for small-scale screening

Yeast Expression:

• Economical eukaryotic system for scale-up

• Capable of many post-translational modifications

• Suitable for stable and durable strains

Mammalian Cell Expression:

• Provides full complement of mammalian post-translational modifications

• Most native-like environment for mammalian proteins

• Often lower yields but highest authenticity

Selection should be based on downstream applications and specific research requirements .

What are the optimal conditions for solubilizing and purifying ZK632.10?

Optimal solubilization and purification of ZK632.10 requires careful consideration of detergents and buffer conditions:

• Solubilization:

  • Dodecyl maltoside (DDM) at 1% concentration has proven effective

  • Solubilization should be performed at 4°C for approximately 1 hour

  • Include benzonase to reduce nucleic acid contamination

• Purification Methods:

  • Immobilized metal affinity chromatography (IMAC) using His-tag

  • Size exclusion chromatography to remove aggregates

  • Buffer conditions: Tris/PBS-based buffer, pH 8.0, with 6% trehalose

• Storage Considerations:

  • Store at -20°C/-80°C upon receipt

  • Aliquoting is necessary to avoid freeze-thaw cycles

  • Optional addition of 50% glycerol for long-term storage

  • Working aliquots can be stored at 4°C for up to one week .

How can researchers optimize small-scale expression screening for ZK632.10 variants?

Small-scale expression screening can be optimized using specialized platforms:

• Equipment Setup:

  • Utilize specialized small-scale eukaryotic expression platforms like Vertiga-IM

  • Culture in 24-well sterile blocks sealed with breathable membranes

  • Maintain shaking at 300 rpm and temperature at 27°C for insect cell cultures

• Infection Protocol:

  • Use mid-log phase insect cell cultures at 2×10^6 cells/ml

  • Infect with high-titer virus stocks (1×10^9 IP/mL) at MOI of 2

  • Maintain cultures for 72 hours post-infection

• Expression Analysis:

  • Monitor cell density and health using microcapillary flow cytometry

  • Evaluate expression by fluorescence using appropriate antibodies

  • Analyze membrane protein extraction using detergent solubilization and immunoblotting

This approach allows efficient parallel screening of multiple protein constructs, saving time and resources .

What techniques are most effective for analyzing ZK632.10 membrane integration?

Several complementary techniques can effectively analyze ZK632.10 membrane integration:

• Biochemical Approaches:

  • Protease protection assays to determine topology

  • Alkylation assays to assess accessibility of cysteine residues

  • Detergent solubility profiles to examine membrane association strength

• Biophysical Methods:

  • Differential scanning calorimetry to assess thermal stability

  • Circular dichroism to analyze secondary structure

  • Fourier-transform infrared spectroscopy for membrane protein structure

• Microscopy Techniques:

  • Confocal microscopy with fluorescent tags to visualize localization

  • Electron microscopy for high-resolution structural information

  • FRET-based approaches to study protein-protein interactions

These methods provide complementary information about how ZK632.10 integrates into membranes and its structural arrangement .

How does ZK632.10 contribute to membrane potential regulation?

Research on PMP3 family proteins indicates that ZK632.10 likely contributes to membrane potential regulation through multiple mechanisms:

• Direct Reciprocal Relationship:

  • Expression levels respond to membrane potential variability (Vmvar)

  • Deletion or knockdown leads to membrane potential changes

  • Functions in a [PMP3(i)hs]-Vmvar regulatory axis

• Ionic Homeostasis:

  • May influence [K+]-Vmvar signaling axis

  • Deletion mutants show conditional growth defects related to ion sensitivity

  • Likely interacts with membrane lipids and/or proteins to modulate ion flux

• Conserved Functions:

  • Cross-kingdom conservation suggests fundamental roles in membrane organization

  • Likely acts in concert with other membrane components

  • Expression levels comparable to housekeeping genes, indicating essential cellular functions

This functional role suggests ZK632.10 is an important regulator of cellular membrane homeostasis .

What protein-protein or protein-lipid interactions have been identified for ZK632.10?

While specific interaction partners for ZK632.10 aren't explicitly detailed in the search results, research on PMP3 family proteins suggests:

• Protein Interactions:

  • Co-localization with other membrane proteins has been observed

  • May interact with ion channels or transporters

  • Potential functional interactions with proteins involved in membrane organization

• Lipid Interactions:

  • Demonstrated lipid-binding selectivity

  • Preference for specific membrane microdomains

  • Interactions likely influenced by the two transmembrane domains

• Research Approaches:

  • Pull-down assays with tagged proteins can identify interaction partners

  • Crosslinking experiments can capture transient interactions

  • Lipidomic analyses can reveal preferred lipid environments

Further research is needed to fully characterize the interactome of ZK632.10, but its membrane localization and functional properties suggest important interactions with both proteins and lipids .

How can researchers accurately assess membrane localization of ZK632.10?

Accurate assessment of ZK632.10 membrane localization can be achieved through multiple complementary approaches:

• Subcellular Fractionation:

  • Differential centrifugation to isolate crude plasma membranes

  • Sucrose gradient ultracentrifugation for membrane purification

  • Detergent fractionation to separate different membrane compartments

• Microscopy-Based Methods:

  • Confocal microscopy with fluorescently tagged protein

  • Immunofluorescence with specific antibodies

  • Super-resolution microscopy for detailed localization

• Biochemical Verification:

  • Western blotting of membrane fractions

  • Protease protection assays to determine topology

  • Surface biotinylation to identify exposed regions

When implementing these techniques, researchers should include appropriate controls such as known plasma membrane markers and intracellular proteins to validate the specificity of localization results .

What approaches are recommended for studying ZK632.10 function through genetic manipulation?

Several genetic manipulation approaches can be employed to study ZK632.10 function:

• Deletion/Knockdown Strategies:

  • CRISPR-Cas9 genome editing for complete gene knockout

  • RNAi for temporary knockdown and dose-dependent analysis

  • Antisense oligonucleotides for targeted suppression

• Expression Modification:

  • Overexpression studies using appropriate vectors

  • Inducible expression systems to control timing and levels

  • Site-directed mutagenesis to create specific functional variants

• Functional Complementation:

  • Cross-species complementation to test functional conservation

  • Expression of eukaryotic homologs in prokaryotic deletion mutants

  • Domain swapping to identify functional regions

• Phenotypic Analysis:

  • Membrane potential measurements to assess electrophysiological changes

  • Salt/stress sensitivity assays to evaluate conditional growth defects

  • Ionic homeostasis measurements to detect altered ion transport

These approaches can reveal how ZK632.10 contributes to membrane potential regulation and cellular physiology under various conditions .

What methods are available for measuring membrane potential changes related to ZK632.10 function?

Several methods can effectively measure membrane potential changes associated with ZK632.10 function:

• Fluorescent Probes:

  • Voltage-sensitive dyes like DiBAC4(3) or JC-1

  • Genetically encoded voltage indicators (GEVIs)

  • Calibration with ionophores for quantitative measurements

• Electrophysiological Techniques:

  • Patch-clamp recordings for direct membrane potential measurement

  • Two-electrode voltage clamp for larger cells

  • Planar patch systems for higher throughput

• Ion-Sensitive Measurements:

  • Fluorescent indicators for relevant ions (K+, Na+, Ca2+)

  • Ion-selective microelectrodes

  • Radioactive ion flux assays

• Experimental Design Considerations:

  • Compare wild-type to deletion/knockdown conditions

  • Assess changes under various stress conditions

  • Combine with pharmacological agents to probe mechanisms

These methods can detect how ZK632.10 influences membrane potential variability (Vmvar) and responds to ionic changes, providing insights into its functional role in membrane homeostasis .

How should researchers interpret contradictory results when studying ZK632.10 in different expression systems?

When encountering contradictory results across different expression systems, researchers should consider:

• System-Specific Factors:

  • Post-translational modifications present in eukaryotic but not prokaryotic systems

  • Membrane composition differences affecting protein folding and function

  • Expression levels varying between systems, potentially causing artifacts

• Methodological Approach:

  • Compare identical constructs across systems (same tags, fusion partners)

  • Validate protein folding and functionality in each system

  • Use multiple independent assays to confirm observations

• Analytical Framework:

  • Consider evolutionary conservation of function versus system-specific adaptations

  • Distinguish between direct protein functions and indirect cellular responses

  • Evaluate if differences reveal mechanistic insights rather than contradictions

• Resolution Strategies:

  • Test functional complementation across systems

  • Create chimeric proteins to identify system-specific domains

  • Use purified protein in defined reconstitution systems to eliminate cellular variables

This systematic approach can help resolve apparent contradictions and provide deeper insights into the protein's true functions across different cellular contexts .

What controls are essential when evaluating membrane potential changes attributed to ZK632.10?

When evaluating membrane potential changes attributed to ZK632.10, several essential controls should be implemented:

• Genetic Controls:

  • Wild-type cells as baseline comparison

  • Complementation with functional protein to rescue phenotypes

  • Expression of non-functional mutants to confirm specificity

  • Empty vector controls for recombinant expression studies

• Experimental Controls:

  • Calibration standards for membrane potential measurements

  • Time-course measurements to distinguish transient from stable effects

  • Membrane integrity verification to exclude non-specific permeabilization

  • Multiple independent measurement techniques to confirm observations

• Environmental Controls:

  • Consistent ionic conditions across experiments

  • Carefully controlled temperature and pH

  • Parallel testing of related PMP3 family members

  • Examination under both standard and stress conditions

• Analytical Controls:

  • Statistical validation of observed differences

  • Dose-response relationships for genetic or pharmacological interventions

  • Consideration of off-target effects from genetic manipulations

  • Correlation of membrane potential changes with other cellular parameters

These comprehensive controls help ensure that observed effects are specifically attributable to ZK632.10 function rather than experimental artifacts or secondary consequences .

How can researchers distinguish between direct and indirect effects of ZK632.10 on cellular physiology?

Distinguishing between direct and indirect effects requires strategic experimental design:

• Temporal Analysis:

  • Immediate responses (seconds to minutes) suggest direct effects

  • Delayed responses (hours to days) may indicate indirect pathways

  • Time-course experiments can reveal sequence of events

• Reconstitution Approaches:

  • Purified protein incorporated into artificial liposomes

  • Minimal systems with defined components

  • Stepwise addition of cellular components to identify dependencies

• Interaction Studies:

  • Direct binding assays with potential partners

  • Proximity labeling to identify molecular neighborhood

  • Pharmacological disruption of specific pathways

• Genetic Strategies:

  • Epistasis analysis with related genes

  • Suppressor screens to identify compensatory pathways

  • Functional domain mapping through mutagenesis

• Comparative Analysis:

  • Cross-species comparison of immediate phenotypes

  • Conservation analysis of interacting partners

  • Correlation between structural features and direct functions

These approaches can help differentiate between the primary actions of ZK632.10 and the downstream consequences that follow from its direct functional role in membrane potential regulation .

How might structural determination of ZK632.10 advance understanding of the UPF0057 family?

Structural determination of ZK632.10 would provide several significant advances:

• Mechanistic Insights:

  • Reveal how this small protein influences membrane potential

  • Identify critical residues for membrane integration

  • Elucidate structural basis for lipid interactions

• Comparative Analysis Opportunities:

  • Provide template for modeling related family members

  • Enable structure-guided functional prediction for uncharacterized homologs

  • Identify conserved structural elements versus variable regions

• Technical Approaches:

  • X-ray crystallography with lipidic cubic phase techniques

  • Cryo-electron microscopy for membrane protein complexes

  • NMR spectroscopy for dynamic structural information

  • Molecular dynamics simulations guided by experimental structures

• Applied Benefits:

  • Guide rational design of mutants for functional studies

  • Inform potential biotechnological applications

  • Enable structure-based understanding of conditional phenotypes

The small size (80 amino acids) makes ZK632.10 potentially amenable to structural studies, though membrane protein crystallization remains challenging and may require innovative approaches .

What is known about the evolutionary conservation of ZK632.10 and how can this inform functional studies?

Evolutionary analysis of ZK632.10 reveals important insights:

• Conservation Patterns:

  • PMP3/UPF0057 family proteins are found across prokaryotes and eukaryotes

  • The functional core (including transmembrane domains) shows high conservation

  • Terminal regions display greater sequence divergence

  • Functional conservation extends across kingdoms despite sequence differences

• Functional Implications:

  • Cross-complementation experiments show functional redundancy

  • Eukaryotic homologues can rescue prokaryotic deletion phenotypes

  • Conservation suggests fundamental roles in membrane biology

  • Expression levels comparable to housekeeping genes indicate essential functions

• Research Applications:

  • Identification of universally conserved residues for targeted mutagenesis

  • Cross-species complementation to map functional domains

  • Comparative analysis of expression patterns and regulation

  • Evolutionary correlation with ionic regulation mechanisms

• Methodological Approaches:

  • Phylogenetic analysis to identify key evolutionary transitions

  • Ancestral sequence reconstruction to test evolutionary hypotheses

  • Correlation of sequence conservation with functional properties

  • Comparison of regulatory mechanisms across diverse species

This evolutionary perspective can guide functional studies by highlighting the most critical elements of ZK632.10 and suggesting experimental approaches based on cross-species comparisons .

What are the implications of ZK632.10 research for understanding membrane homeostasis in higher organisms?

Research on ZK632.10 has broader implications for understanding membrane homeostasis:

• Fundamental Mechanisms:

  • The [PMP3(i)hs]-Vmvar axis represents a key element in membrane homeostasis

  • Small membrane proteins can have outsized effects on cellular physiology

  • Membrane potential regulation involves complex protein-lipid interactions

  • Evolutionary conservation indicates fundamental cellular processes

• Biomedical Relevance:

  • Membrane potential dysregulation is implicated in numerous pathologies

  • Homologous proteins in higher organisms may serve similar functions

  • Understanding basic mechanisms can inform therapeutic approaches

  • Potential relevance to excitable cell function in neural and cardiac systems

• Research Directions:

  • Identification and characterization of mammalian homologs

  • Investigation of roles in specialized cell types with critical membrane functions

  • Exploration of potential involvement in disease-related membrane dysfunction

  • Integration with broader ionic homeostasis mechanisms

• Technical Applications:

  • Development of membrane potential biosensors based on functional mechanisms

  • Engineering of membrane proteins with tailored properties

  • Application to biotechnology for controlled membrane permeability

  • Potential model systems for studying membrane protein dynamics

These broader implications highlight the importance of research on seemingly simple proteins like ZK632.10 for understanding fundamental aspects of cellular physiology across species .

What are the best storage and handling practices for recombinant ZK632.10?

To maintain optimal activity of recombinant ZK632.10, follow these storage and handling guidelines:

• Initial Processing:

  • Briefly centrifuge vial before opening to bring contents to bottom

  • Reconstitute lyophilized protein in deionized sterile water (0.1-1.0 mg/mL)

  • Add glycerol to 5-50% final concentration for long-term storage (50% recommended)

• Storage Conditions:

  • Store at -20°C/-80°C for long-term preservation

  • Aliquot to avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

• Critical Precautions:

  • Avoid repeated freezing and thawing

  • Maintain cold chain during handling

  • Minimize exposure to detergents unless during intentional solubilization

  • Use appropriate buffer systems (Tris/PBS-based buffer, pH 8.0)

• Quality Control Measures:

  • Verify purity by SDS-PAGE before experiments (should be >90%)

  • Monitor stability over time with activity assays

  • Consider fresh preparations for critical experiments

These practices will help maintain the structural integrity and functional properties of the recombinant protein throughout the research process .

What concentration determination methods are most accurate for ZK632.10?

Several methods can be used to determine ZK632.10 concentration, each with advantages:

• Absorbance-Based Methods:

  • UV absorbance at 280 nm using calculated extinction coefficient

  • Adjust for interference from buffer components

  • Requires pure protein preparations

• Colorimetric Assays:

  • BCA protein assay (most recommended for membrane proteins)

  • Bradford assay (less ideal due to detergent interference)

  • Modified Lowry method (suitable with detergent corrections)

• Advanced Techniques:

  • Amino acid analysis for absolute quantification

  • Quantitative mass spectrometry with internal standards

  • Fluorescence-based quantification with appropriate tags

• Practical Implementation:

  • Use bovine serum albumin as reference standard

  • Construct standard curves in identical buffer conditions

  • Perform measurements in triplicate

  • Account for detergent effects on readings

The BCA protein assay has been successfully used for ZK632.10 and related membrane proteins, offering good compatibility with common detergents used in membrane protein research .

What troubleshooting approaches are recommended when ZK632.10 expression yields are low?

When faced with low expression yields of ZK632.10, consider these troubleshooting strategies:

• Expression System Optimization:

  • Test alternative expression systems (E. coli, insect cells, yeast)

  • Optimize codon usage for the expression host

  • Evaluate different fusion tags (His, GST, MBP)

  • Test induction conditions (temperature, time, inducer concentration)

• Construct Design Refinement:

  • Generate truncation variants to identify stable domains

  • Modify N- or C-terminal regions that may affect expression

  • Consider fusion proteins known to enhance membrane protein expression

  • Test different signal sequences for proper membrane targeting

• Culture Condition Adjustments:

  • Reduce expression temperature to slow folding

  • Add specific lipids to culture medium

  • Supplement with osmolytes or chemical chaperones

  • Adjust media composition for optimal cell health

• Analytical Approaches:

  • Quantify mRNA levels to identify transcriptional limitations

  • Assess protein degradation during expression

  • Examine toxic effects on host cells

  • Monitor protein localization to identify improper targeting

This systematic approach to troubleshooting can help identify and overcome specific bottlenecks in ZK632.10 expression, leading to improved yields for downstream applications .