Recombinant Mouse Protein Asterix (Wdr83os)

What are the common synonyms and identifiers for Mouse Protein Asterix?

When searching literature or databases, researchers should be aware of these alternative names:

• Wdr83os (primary gene name)

• PAT complex subunit Asterix

• Protein WDR83OS homolog

• Protein associated with the ER translocon of 10kDa (PAT-10)

• PAT10

The UniProt ID for Mouse Protein Asterix is Q6ZWX0 .

How is Wdr83os conserved across species?

Wdr83os is highly conserved across multiple species. Orthologs have been identified in humans (C19orf56), mouse, rat, bovine, frog, zebrafish, and chimpanzee . This high degree of conservation suggests crucial functional roles in cellular processes. Comparative studies have shown that the protein maintains its core functional domains across these species, with sequence homology particularly preserved in the regions associated with translocon complex interaction.

What are the optimal conditions for expressing Recombinant Mouse Protein Asterix in E. coli?

For optimal expression of Recombinant Mouse Protein Asterix in E. coli, researchers should consider:

• Expression System: BL21(DE3) strain is commonly used for high-yield expression

• Expression Vector: pET vectors with T7 promoter systems show good results

• Induction Parameters:

  • IPTG concentration: 0.5-1.0 mM

  • Induction temperature: 16-18°C (lower temperatures reduce inclusion body formation)

  • Induction duration: 16-20 hours

• Media: Enriched media such as 2XYT or TB can increase yield

The accessibility of translation initiation sites has been identified as the strongest predictor of heterologous protein expression in E. coli . Poor expression levels are often associated with stable mRNA structures that impede ribosome assembly and progress, particularly around the Shine-Dalgarno sequence and translation initiation site .

What purification strategy yields the highest purity for Recombinant Mouse Protein Asterix?

A multi-step purification strategy is recommended:

• Initial Capture: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with gradual imidazole elution (20-250 mM)

• Intermediate Purification: Size exclusion chromatography to separate target protein from aggregates and other impurities

• Polishing Step: Ion exchange chromatography (typically anion exchange) based on the protein's theoretical pI

• Quality Control: SDS-PAGE analysis should confirm purity >90%

Post-purification, the protein is typically obtained as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

How can I optimize the stability of purified Recombinant Mouse Protein Asterix?

To maximize stability and activity:

• Storage Buffer Optimization:

  • Tris/PBS-based buffer with 6% trehalose at pH 8.0 has shown good results

  • Addition of 50% glycerol significantly improves long-term stability

• Storage Conditions:

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

  • Avoid repeated freeze-thaw cycles (prepare working aliquots)

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

• Reconstitution Protocol:

  • Briefly centrifuge the vial before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration before aliquoting

How does Wdr83os function in the multi-pass translocon (MPT) complex?

As a component of the MPT complex, Wdr83os (Asterix) plays a critical role in membrane protein biogenesis. The protein functions as:

• Assembly Factor: Assists in the formation and stability of the MPT complex

• Substrate Recognition: Helps identify multi-pass membrane proteins that require insertion

• ER Membrane Integration: Facilitates the correct orientation and insertion of transmembrane segments

Research shows that Wdr83os depletion results in significant defects in the insertion of multi-pass membrane proteins, particularly those with complex topology. This suggests it may play a specialized role in handling challenging membrane protein clients compared to single-pass membrane proteins.

What experimental approaches are most effective for studying Wdr83os interactions with other components of the translocon?

Several complementary techniques have proven effective:

• Co-immunoprecipitation (Co-IP): Using anti-Wdr83os antibodies to pull down interaction partners

• Proximity Labeling: BioID or APEX2 fusion constructs to identify proximal proteins in living cells

• Crosslinking Mass Spectrometry: To capture transient interactions within the MPT complex

• FRET/BRET Assays: For monitoring real-time interactions in living cells

• Cryo-EM Analysis: For structural determination of Wdr83os within the MPT complex

These methods have revealed that Wdr83os interacts with multiple components of the ER translocon machinery, including Sec61 complex members and other PAT complex proteins.

What is known about the tissue-specific expression patterns of Wdr83os in mouse models?

Wdr83os exhibits a widespread but variable expression pattern across mouse tissues:

• High Expression:

  • Brain (particularly in neurons)

  • Liver

  • Kidney

  • Pancreas

• Moderate Expression:

  • Heart

  • Lung

  • Intestine

• Low Expression:

  • Skeletal muscle

  • Adipose tissue

How can I design loss-of-function experiments to study Wdr83os function in membrane protein biogenesis?

For effective loss-of-function studies:

• siRNA/shRNA Approaches:

  • Target sequences showing highest knockdown efficiency:

    • siRNA1: 5'-GAUCCAUGACUACAUGUAU-3'

    • siRNA2: 5'-AGCUCGAACCUGCAAGGAU-3'

  • Validate knockdown by qRT-PCR and Western blot

  • Allow 48-72 hours for effective protein depletion

• CRISPR/Cas9 Knockout:

  • Guide RNA target sites in early exons:

    • gRNA1: 5'-CGUACCCACCUCCGAGUGCG-3'

    • gRNA2: 5'-AGUGGCAUGUGUAUUGAGCA-3'

  • Consider inducible systems for complete knockout may be lethal

• Phenotypic Analysis:

  • Monitor accumulation of unfolded proteins in the ER

  • Assess membrane protein insertion using topological reporters

  • Evaluate ER stress markers (XBP1 splicing, ATF6 cleavage, PERK phosphorylation)

What are the best reporter systems to study Wdr83os-dependent membrane protein insertion?

Several reporter systems have proven valuable:

• Split GFP Complementation Assay:

  • One GFP fragment fused to Wdr83os

  • Complementary fragment attached to candidate substrate proteins

  • Fluorescence indicates successful interaction

• Glycosylation Site Insertion:

  • Strategic placement of N-X-S/T sequences in substrate proteins

  • Glycosylation occurs only on ER-lumenal domains

  • Mobility shift on SDS-PAGE indicates successful membrane insertion

• Protease Protection Assay:

  • Microsomes containing tagged substrate proteins

  • Protease digestion cleaves only cytosolic domains

  • Western blot analysis reveals membrane topology

• Bioluminescence Resonance Energy Transfer (BRET):

  • RLuc8-tagged Wdr83os and Venus-tagged substrate

  • Real-time monitoring of interaction dynamics during membrane insertion

How can I analyze the effect of Wdr83os mutations on its function?

To systematically analyze Wdr83os mutations:

• Structure-Function Analysis:

  • Create alanine scanning libraries across the protein sequence

  • Focus on conserved residues or predicted functional domains

  • Express in Wdr83os-depleted cells and assess rescue capability

• Domain Swapping:

  • Exchange domains with orthologs from other species

  • Create chimeric proteins to map essential functional regions

  • Identify species-specific functions

• Post-translational Modification Analysis:

  • Identify and mutate phosphorylation, ubiquitination, or other PTM sites

  • Monitor effects on protein stability, localization, and function

  • Use phospho-mimetic and phospho-dead mutations to assess regulation

• Readout Systems:

  • Membrane protein reporter insertion efficiency

  • Co-immunoprecipitation with known interaction partners

  • Subcellular localization by immunofluorescence microscopy

What are the common challenges in working with Recombinant Mouse Protein Asterix and how can they be overcome?

How can I validate that my recombinant Wdr83os protein is properly folded and functional?

To confirm proper folding and functionality:

• Structural Analysis:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Limited proteolysis pattern comparison with native protein

  • Thermal shift assay to measure stability and ligand binding

• Functional Assays:

  • In vitro membrane protein insertion assay using purified components

  • Rescue experiments in Wdr83os-depleted cells

  • Co-immunoprecipitation with known binding partners

• Biophysical Characterization:

  • Size exclusion chromatography to confirm monomeric state

  • Dynamic light scattering to assess homogeneity

  • Intrinsic tryptophan fluorescence to monitor tertiary structure

What controls should be included when studying Wdr83os-dependent membrane protein insertion?

Essential controls include:

• Positive Controls:

  • Well-characterized Wdr83os-dependent multi-pass membrane proteins

  • PAT complex-dependent substrates (e.g., G protein-coupled receptors)

• Negative Controls:

  • Single-pass membrane proteins that use alternative insertion pathways

  • Cytosolic proteins that should not interact with Wdr83os

  • Secretory proteins that use the Sec61 translocon but not the PAT complex

• System Validation Controls:

  • Sec61 inhibition (using cotransin) should block all membrane protein insertion

  • General translation inhibition (using cycloheximide) versus pathway-specific effects

  • Rescue experiments with wild-type Wdr83os in knockout/knockdown systems