Recombinant Xenopus laevis Chloride channel CLIC-like protein 1 (clcc1)

Where is CLCC1 primarily localized in cells and what is its proposed function?

CLCC1 is predominantly localized to the endoplasmic reticulum (ER). Biochemical analyses and immunofluorescence microscopy confirm this localization through:

• Co-localization with ER markers like CALNEXIN

• Absence of significant co-localization with Golgi, lysosomal, or plasma membrane markers

CLCC1's proposed functions include:

• Maintenance of ER ion homeostasis

• Regulation of the unfolded protein response (UPR)

• Membrane fusion during nuclear envelope reorganization

• Lipid bilayer equilibration in collaboration with scramblases like TMEM41B

• Prevention of ER stress-induced cell death

These functions position CLCC1 as a critical mediator of ER homeostasis and cellular stress responses .

How does CLCC1 differ between species and what is unique about Xenopus laevis CLCC1?

CLCC1 is highly conserved across vertebrates but with notable differences:

Unique to Xenopus laevis CLCC1:

• Critical role in fertilization membrane development

• Higher expression in oocytes compared to other species

• Distinctive intracellular distribution pattern during early development

• Different electrophysiological properties compared to mammalian homologs

What are the optimal conditions for expressing recombinant Xenopus laevis CLCC1?

Successful expression of recombinant Xenopus laevis CLCC1 requires careful optimization:

Expression Systems Comparison:

Recommended Protocol:

• Mammalian Expression (HEK293):

  • Transfect cells with CLCC1 cDNA in appropriate vector (pCMV or pEF1α)

  • Harvest cells after 48-72 hours

  • Solubilize membranes with 1% Triton X-100 in 20 mM Tris pH 7.5, 150 mM NaCl with protease inhibitors

  • Purify using affinity chromatography

• E. coli Expression and Refolding:

  • Express in inclusion bodies

  • Solubilize in 8M urea or 6M guanidine HCl

  • Refold by rapid dilution into buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM CaCl₂, and 0.1% CHAPS

  • Gradually remove denaturant by dialysis

The choice of expression system depends on the experimental requirements and available resources .

What affinity tags and purification strategies work best for recombinant Xenopus laevis CLCC1?

Various affinity tags have been successfully used for CLCC1 purification, each with distinct advantages:

Tag Comparison for CLCC1 Purification:

Recommended Purification Protocol:

• Two-step purification for highest purity:

  • Primary affinity chromatography (based on tag)

  • Size exclusion chromatography using Superdex 200 in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT

• For functional studies:

  • Consider using smaller tags (His₆ or FLAG)

  • Verify protein integrity by gel filtration profile (predominant tetrameric form)

  • Test activity in reconstituted liposomes

• Storage conditions:

  • 50 mM Tris-based buffer with 50% glycerol at -20°C for short-term

  • Aliquot and store at -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles

The GST-tagged CLCC1 has shown good stability and retention of function, making it suitable for many applications, while His-tagged versions allow for more compact protein preparation when size is a concern .

How can I verify the proper folding and activity of purified recombinant Xenopus laevis CLCC1?

Multiple complementary approaches should be used to verify proper folding and activity:

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy:

  • Secondary structure content should show typical α-helical pattern (minima at 208 and 222 nm)

  • Thermal stability with melting temperature around 48-52°C

• Size Exclusion Chromatography:

  • Properly folded CLCC1 elutes primarily as a tetramer (~220 kDa with tags)

  • Misfolded protein shows aggregation in the void volume

• Limited Proteolysis:

  • Correctly folded protein shows resistance to limited trypsin digestion

  • Characteristic fragmentation pattern with major fragments at ~35 and ~25 kDa

Functional Validation Methods:

• Liposome Reconstitution Assay:

  • Incorporate purified CLCC1 into liposomes containing chloride-sensitive fluorophores

  • Measure chloride flux upon application of external chloride gradient

  • Expected EC₅₀ for chloride transport: 40-70 nM for properly folded protein

• Calcium-Binding Assessment:

  • Monitor structural changes by intrinsic tryptophan fluorescence upon Ca²⁺ binding

  • Properly folded protein shows Kd values of 0.2-0.5 μM for calcium

• Immunoprecipitation with Known Partners:

  • Verify interaction with known binding partners (e.g., viral proteins like ORF3A)

  • Co-immunoprecipitation should pull down partner proteins in expected stoichiometry

Properly folded recombinant CLCC1 should demonstrate both the expected structural characteristics and functional activity in at least two independent assays .

How can recombinant Xenopus laevis CLCC1 be used to study viral interactions and pathogenesis?

Recombinant CLCC1 serves as a valuable tool for investigating viral interactions, particularly with herpesviruses and coronaviruses:

SARS-CoV-2 Interaction Studies:

• Co-immunoprecipitation protocol:

  • Express recombinant CLCC1-FLAG and SARS-CoV-2 ORF3A-HA in HEK293T cells

  • Lyse cells in buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease inhibitors

  • Immunoprecipitate using anti-FLAG antibody pre-bound to protein G beads

  • Wash four times with lysis buffer

  • Elute with 1× LDS sample buffer and analyze by western blot

  • Expected result: ORF3A co-precipitates with CLCC1

• Fluorescence microscopy co-localization:

  • Co-express fluorescently-tagged CLCC1 and ORF3A

  • Fix cells and stain for ER markers

  • Analyze co-localization using confocal microscopy

  • Expected observation: Partial co-localization at ER membranes

Herpesvirus Studies:

• Nuclear egress analysis:

  • Express recombinant CLCC1 in CLCC1-knockout cells infected with herpesvirus

  • Monitor capsid localization by electron microscopy or fluorescence microscopy

  • Quantify nuclear vs. cytoplasmic capsids

  • Expected result: CLCC1 expression rescues nuclear egress defect observed in knockout cells

• Viral titer restoration:

  • Complement CLCC1-knockout cells with CLCC1 variants

  • Infect with herpesvirus and measure viral titers at multiple timepoints

  • Expected result: Wild-type CLCC1 restores viral titers by approximately 1000-fold compared to knockout

The recombinant protein can be used to define the binding interface between CLCC1 and viral proteins through mutagenesis studies, potentially revealing new antiviral targets .

What role does recombinant CLCC1 play in studying ER stress and the unfolded protein response?

Recombinant CLCC1 is a powerful tool for investigating ER stress mechanisms:

UPR Activation Studies:

• Gene expression analysis protocol:

  • Express wild-type or mutant CLCC1 in cells

  • Extract RNA and perform RT-qPCR or RNA-seq

  • Measure expression of UPR markers: HSPA6, HERPUD1, XBP1 splicing, CHOP

  • Expected results: Wild-type CLCC1 maintains normal UPR, while mutants may activate UPR

• XBP1 splicing quantification:

  • Increased splicing indicates UPR activation

Cell Death Protection Assays:

• ER stress induction:

  • Treat cells with tunicamycin (2-5 μg/ml) or thapsigargin (0.5-1 μM)

  • Express recombinant CLCC1 before treatment

  • Measure cell viability using MTT or annexin V/PI staining

  • Expected result: CLCC1 expression protects against ER stress-induced death

• Rescue of mutant phenotypes:

  • Express CLCC1 in cells treated with TUDCA (taurodeoxycholic acid, 500 μM)

  • Assess cell viability and UPR markers

  • Expected result: TUDCA treatment mimics CLCC1 protective effects

CLCC1 and UPR Gene Regulation:

These studies revealed that CLCC1 serves as a critical regulator of ER homeostasis and stress responses .

How can recombinant CLCC1 be used in membrane biology and lipid transport studies?

Recombinant CLCC1 provides valuable insights into membrane biology:

Liposome-Based Assays:

• Lipid scrambling analysis protocol:

  • Prepare liposomes with fluorescently-labeled phospholipids (NBD-PC)

  • Incorporate recombinant CLCC1 alone or in complex with TMEM41B

  • Add dithionite to quench NBD-PC signals at the outer leaflet

  • Monitor fluorescence decrease over time

  • Expected result: CLCC1 alone shows minimal scrambling activity, but enhances TMEM41B-mediated scrambling

• Leakage control experiments:

  • Encapsulate self-quenching fluorescent dye (calcein) in liposomes

  • Monitor fluorescence increase due to potential leakage

  • Expected result: No significant leakage in CLCC1-containing liposomes

Bilayer Equilibration Assays:

• ER membrane asymmetry analysis:

  • Express recombinant tagged CLCC1 in cells

  • Isolate ER membranes and analyze lipid distribution between leaflets

  • Expected result: CLCC1 promotes equilibration of phospholipids between leaflets

Quantitative Data on CLCC1-Enhanced Lipid Scrambling:

These experiments demonstrate that CLCC1 substantially enhances TMEM41B scramblase activity without compromising membrane integrity, suggesting a regulatory role in lipid homeostasis .

How can structure-function studies of CLCC1 be designed using recombinant protein variants?

Advanced structure-function analyses of CLCC1 require strategic mutation design:

Critical Domains for Mutation Analysis:

• Transmembrane domains:

  • TM1 (aa 180-200): Contains conserved charged residues important for ion selectivity

  • TM2 (aa 220-240): Forms part of the pore structure

  • TM3 (aa 260-280): Contains residues for oligomerization

• Disease-associated mutations:

  • D25E: Associated with retinitis pigmentosa, affects channel function while maintaining protein-protein interactions

  • Other potential sites based on human variants: R196Q, G345S, L434P

Experimental Design for Functional Assessment:

• Electrophysiological characterization:

  • Express CLCC1 variants in Xenopus oocytes or reconstitute in planar lipid bilayers

  • Measure chloride conductance using two-electrode voltage clamp or patch clamp

  • Parameters to assess: conductance, ion selectivity, voltage dependence, pharmacology

  • Expected results: D25E mutant shows ~60% reduced chloride conductance compared to wild-type

• Oligomerization analysis:

  • Perform blue native PAGE and crosslinking studies with purified proteins

  • Compare wild-type vs. mutant CLCC1

  • Expected result: Wild-type forms tetramers, while specific TM3 mutations may disrupt oligomerization

• Reconstitution system for ion flux measurement:

  • Reconstitute purified CLCC1 variants into liposomes loaded with chloride-sensitive fluorophores

  • Monitor chloride flux in response to gradients

  • Determine relative transport rates of variants

Comprehensive Mutation Analysis Strategy:

This systematic approach will help define the structure-function relationships in CLCC1 and may provide insights for therapeutic targeting .

What are the approaches for studying CLCC1's role in development using recombinant protein?

Recombinant CLCC1 enables detailed investigation of its developmental functions:

Xenopus Development Studies:

• Microinjection rescue experiments:

  • Deplete endogenous CLCC1 using morpholinos or CRISPR

  • Microinject recombinant CLCC1 protein or mRNA encoding wild-type or mutant CLCC1

  • Assess developmental phenotypes and fertilization membrane formation

  • Expected result: Wild-type CLCC1 rescues developmental defects, while D25E mutant shows partial rescue

• Fertilization assays:

  • Treat Xenopus eggs with neutralizing antibodies against CLCC1

  • Add recombinant CLCC1 to rescue fertilization membrane formation

  • Quantify fertilization rate and polyspermy frequency

  • Expected result: Recombinant CLCC1 restores normal fertilization rate

Zebrafish Models:

• TALEN knockout rescue protocol:

  • Generate clcc1-/- zebrafish using TALENs (targeting exon 1)

  • Inject recombinant CLCC1 protein or mRNA

  • Assess survival, eye development, and retinal structure

  • Expected rescue effects:

    • Prevents lethality at 11 dpf

    • Restores retinal thickness

    • Normalizes opsin expression

Quantitative Rescue Data in Zebrafish Models:

These rescue experiments demonstrate the critical function of CLCC1 in vertebrate development and provide models for testing structure-function relationships in vivo .

What are the most promising therapeutic applications for recombinant CLCC1 or its modifiers?

Recombinant CLCC1 research points to several therapeutic applications:

Retinal Degeneration Therapy:

• Gene therapy approaches:

  • AAV-mediated delivery of wild-type CLCC1 to retinal cells

  • Rescue of D25E mutation-associated retinitis pigmentosa

  • Preclinical results in mouse models show:

    • Preservation of photoreceptor layer thickness

    • Improved ERG responses

    • Extended survival of photoreceptors

• Small molecule screening:

  • Use recombinant CLCC1 for high-throughput screening

  • Identify compounds that enhance mutant CLCC1 function

  • Chemical chaperones like TUDCA show promise in preliminary studies

Viral Infection Interventions:

• Interaction inhibitor development:

  • Screen peptides or small molecules that disrupt CLCC1-viral protein interactions

  • Target the binding interface between CLCC1 and SARS-CoV-2 ORF3A

  • Potential reduction in virus-induced cytotoxicity

• UPR modulation strategies:

  • Develop compounds that mimic CLCC1's protective effect against ER stress

  • Focus on downstream UPR modulators like HSPA6 and HERPUD1

  • Potential therapeutic window between homeostatic and terminal UPR

Promising Compounds for CLCC1 Modulation:

These therapeutic approaches leverage CLCC1's pivotal role in ER homeostasis, making it a promising target for diseases involving ER stress and viral infections .

How can I overcome aggregation issues when working with recombinant Xenopus laevis CLCC1?

Membrane protein aggregation is a common challenge with CLCC1:

Prevention Strategies:

• Optimized buffer conditions:

  • Maintain pH between 7.2-7.8 (optimal: 7.5)

  • Include glycerol (10-20%) to stabilize native structure

  • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol)

  • Include mild detergents (0.1% CHAPS or 0.03% DDM)

• Solubilization approaches:

  • Mild detergent panel screening:

  Detergent	Concentration	Solubilization Efficiency	Effect on Activity
  DDM	0.03-0.1%	75-85%	Maintains activity
  CHAPS	0.5-1.0%	60-70%	Maintains activity
  Triton X-100	0.1-0.5%	80-90%	Partial reduction
  Digitonin	0.5-1.0%	65-75%	Maintains activity
  FC-12	0.1-0.3%	90-95%	Significant reduction

• Fusion partners for improved solubility:

  • MBP tag increases solubility by ~60%

  • SUMO tag improves yield and reduces aggregation

  • TrxA tag provides moderate improvement in solubility

Rescue Strategies for Aggregated Protein:

• Mild denaturation and refolding:

  • Solubilize aggregates with 2M urea

  • Gradually remove urea by dialysis against buffer containing detergent

  • Recovery efficiency: 30-40% of functional protein

• Size exclusion chromatography:

  • Separate aggregates using Superose 6 column

  • Collect monomeric and tetrameric fractions

  • Concentrate using 100 kDa MWCO concentrators

Analytical Techniques to Monitor Aggregation:

• Dynamic light scattering (DLS) to detect early aggregation

• Analytical ultracentrifugation to characterize oligomeric state

• Thermal shift assays to identify stabilizing conditions

When working with CLCC1, maintain the protein at concentrations below 2 mg/ml and perform all manipulations at 4°C to minimize aggregation .

What controls and validation steps are critical when studying CLCC1 interactions with viral proteins?

Robust controls and validation are essential for CLCC1-viral protein interaction studies:

Essential Controls for Co-immunoprecipitation:

• Negative controls:

  • Non-binding mutant CLCC1 (e.g., specific C-terminal truncation)

  • Unrelated membrane protein of similar topology

  • Empty vector expression

  • Isotype-matched control antibody for immunoprecipitation

• Positive controls:

  • Known CLCC1 interaction partner (e.g., TMEM41B)

  • Previously validated viral protein interaction

  • Direct pull-down with purified proteins

Validation Hierarchy:

• Primary detection: Co-immunoprecipitation with antibodies against endogenous proteins

• Orthogonal validation: Proximity ligation assay (PLA) or FRET between tagged proteins

• Functional validation: Mutational analysis affecting interaction but not individual protein stability

• Direct binding: Surface plasmon resonance or microscale thermophoresis with purified components

• Structural validation: Crosslinking mass spectrometry or cryo-EM of complex

Critical Parameters to Control:

Statistical analysis approach:

• Perform at least three independent biological replicates

• Normalize to input levels for each protein

• Apply appropriate statistical tests (t-test or ANOVA)

• Report effect size and confidence intervals

Following this validation framework ensures reliable identification of genuine CLCC1-viral protein interactions and minimizes false positives .

How can I troubleshoot activity measurement issues with recombinant CLCC1 in functional assays?

Activity measurement challenges with CLCC1 can be addressed systematically:

Liposome-Based Activity Assay Troubleshooting:

• Low/no activity detected:

  • Potential causes:

    • Improper protein orientation in liposomes

    • Inactive protein due to purification conditions

    • Suboptimal buffer composition

  • Solutions:

    • Verify protein incorporation by fluorescent labeling or western blot

    • Add 0.1% cholesterol to liposomes to promote proper insertion

    • Optimize reconstitution by testing protein:lipid ratios (1:100 to 1:1000)

    • Include 5 mM calcium in buffer (critical for activity)

• High background or leaky liposomes:

  • Potential causes:

    • Detergent contamination

    • Osmotic imbalance

    • Liposome instability

  • Solutions:

    • Extend detergent removal time with Bio-Beads

    • Perform calcein leakage assay as control

    • Use more stable lipid compositions (include 10% DOPE)

    • Verify liposome integrity by dynamic light scattering

Cell-Based Assay Troubleshooting:

• Expression verification:

  • Confirm proper localization using immunofluorescence

  • Verify expression levels by western blot

  • Use surface biotinylation to assess membrane integration

• Functional readouts optimization:

  • For chloride flux: Calibrate MQAE fluorescence with known chloride standards

  • For ER stress: Include positive controls (tunicamycin, thapsigargin)

  • For cell viability: Test multiple assays (MTT, LDH release, Annexin V/PI)

Quantitative Troubleshooting Guidance:

For maximum reproducibility, prepare large batches of liposomes, aliquot and freeze at -80°C, and use consistent protein:lipid ratios across experiments .