Recombinant Mouse UPF0766 protein C6orf228 homolog

What is the UPF0766 protein C6orf228 homolog in mice?

UPF0766 protein C6orf228 homolog is a small integral membrane protein (also known as Smim13) consisting of 88 amino acids. It belongs to an uncharacterized protein family (UPF) with the complete amino acid sequence: MWHNVGLTLLVFVATLLIVLLLMVCGWYFVWHLFLSKFKFLRELVGDTGSQEGDNEQPSGSETEEDPSASPQKIRSARQRRPPVDAGH. The protein is encoded by the Smim13 gene and has been assigned the UniProt ID E9Q942 .

What is the structural classification of mouse UPF0766 protein?

Based on sequence analysis, mouse UPF0766 is classified as a transmembrane protein with hydrophobic regions consistent with membrane integration. The N-terminal region (approximately amino acids 1-30) shows a characteristic pattern of a transmembrane domain with multiple hydrophobic residues. The protein lacks well-characterized functional domains but contains potential phosphorylation sites in the C-terminal region .

What are the recommended storage conditions for recombinant mouse UPF0766 protein?

The recombinant UPF0766 protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple uses. Working aliquots can be stored at 4°C for up to one week. The protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose (pH 8.0). Repeated freeze-thaw cycles should be avoided as they may compromise protein stability and activity .

What reconstitution methods are recommended for lyophilized recombinant UPF0766 protein?

The recommended protocol for reconstitution involves briefly centrifuging the vial before opening to bring contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, adding glycerol to a final concentration of 5-50% (with 50% being standard) and aliquoting before storage at -20°C/-80°C is advised .

What are the key experimental design principles when studying an uncharacterized protein like UPF0766?

When studying uncharacterized proteins like UPF0766, implement a systematic experimental approach:

• Begin with bioinformatic analysis to predict properties and potential functions

• Design experiments with proper controls, including:

  • Positive and negative controls

  • Technical replicates (minimum of three)

  • Biological replicates (different protein preparations)

• Use multiple complementary techniques to verify findings

• Include concentration gradients when testing protein effects

• Establish clear, testable hypotheses with specific variables

How can proteomics approaches be used to investigate the function of UPF0766 protein?

Proteomics can be leveraged to investigate UPF0766 through:

• Co-immunoprecipitation studies - Use anti-His antibodies to pull down the recombinant protein along with potential binding partners from cell lysates

• Proximity labeling - Fuse UPF0766 with enzymes like BioID or APEX2 to identify proteins in close proximity

• Comparative proteomics - Compare proteome profiles between wild-type cells and those overexpressing or lacking UPF0766

• Post-translational modification analysis - Identify potential phosphorylation, glycosylation, or other modifications

Mass spectrometry analysis should follow standardized protocols similar to those used in kidney tissue proteomic studies, where proteins were classified by localization and function .

What expression systems are suitable for producing recombinant mouse UPF0766 protein?

Based on the available data, the following expression systems can be considered:

The E. coli system has been successfully used for commercial production, suggesting it provides functional protein despite being a prokaryotic system .

What purification strategies are most effective for recombinant His-tagged UPF0766 protein?

For optimal purification of His-tagged UPF0766 protein:

• Cell lysis optimization:

  • For E. coli-expressed protein: Use mild detergents (0.5-1% Triton X-100 or NP-40) in lysis buffer

  • Include protease inhibitors to prevent degradation

• Affinity chromatography:

  • Ni-NTA resin is preferred for His-tagged proteins

  • Optimize imidazole concentration in binding buffer (10-20 mM) to reduce non-specific binding

  • Use step gradient elution (50-250 mM imidazole)

• Secondary purification:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Ion exchange chromatography as a polishing step

• Quality control:

  • SDS-PAGE with silver staining to assess >90% purity

  • Western blot to confirm identity

  • Dynamic light scattering to evaluate homogeneity

This approach follows principles similar to those used for other His-tagged recombinant proteins .

How can researchers investigate potential interaction partners of UPF0766 protein?

To investigate potential interaction partners:

• Yeast two-hybrid screening:

  • Clone UPF0766 as bait construct

  • Screen against mouse tissue-specific libraries (particularly kidney-derived)

  • Verify interactions with co-immunoprecipitation

• Proximity-based labeling:

  • Generate fusion proteins with BioID or APEX2

  • Express in relevant cell lines

  • Identify biotinylated proteins by mass spectrometry

• Surface plasmon resonance (SPR):

  • Immobilize purified UPF0766 on sensor chip

  • Test candidate interactors in concentration series

  • Determine binding kinetics and affinity constants

• Crosslinking mass spectrometry:

  • Use membrane-permeable crosslinkers

  • Identify crosslinked peptides by specialized MS/MS analysis

  • Map interaction interfaces

This multi-method approach provides complementary evidence for protein interactions .

What strategies can be employed to study the membrane topology of UPF0766 protein?

To characterize the membrane topology of UPF0766:

• Protease protection assays:

  • Express UPF0766 in microsomes or membrane vesicles

  • Treat with proteases with/without membrane permeabilization

  • Analyze protected fragments by Western blotting

• Fluorescence microscopy with epitope tags:

  • Generate constructs with fluorescent tags at N- and C-termini

  • Use selective permeabilization with digitonin vs. Triton X-100

  • Determine which epitopes are accessible under different conditions

• Glycosylation mapping:

  • Introduce artificial N-glycosylation sites at various positions

  • Express in glycosylation-competent systems

  • Identify glycosylated sites as extracellular/luminal domains

• FRET-based approaches:

  • Create donor-acceptor pairs at predicted membrane interfaces

  • Measure energy transfer efficiency

  • Map relative positions of protein segments

These techniques provide complementary data about membrane protein orientation .

How does UPF0766 protein compare to other small integral membrane proteins in terms of research approaches?

Comparison of research approaches between UPF0766 and other small integral membrane proteins:

This comparative analysis highlights the need for specialized approaches when working with membrane proteins like UPF0766.

What insights from peroxisomal protein research might be applicable to studying UPF0766?

Research methodologies from peroxisomal protein studies that could be applied to UPF0766 investigation:

• Subcellular fractionation techniques:

  • Density gradient centrifugation to determine precise localization

  • Comparative analysis with known organelle markers

• Metabolic function analysis:

  • Metabolomic profiling in knockdown/knockout models

  • Substrate utilization assays if enzymatic activity is suspected

• Protein complex characterization:

  • Blue native PAGE to identify native complexes

  • Chemical crosslinking followed by MS analysis

• Disease association studies:

  • Examine expression in disease models (especially kidney disorders)

  • Analyze correlation with other disease-associated proteins

Several peroxisomal proteins showed differential expression in diabetic mouse kidney models, suggesting involvement in metabolic regulation that might also apply to UPF0766 .

What are common challenges in working with recombinant membrane proteins like UPF0766 and how can they be addressed?

Common challenges and solutions for recombinant membrane protein work:

• Low expression yields:

  • Solution: Optimize codon usage for expression system

  • Solution: Test multiple promoters and expression conditions

  • Solution: Consider fusion partners that enhance expression (SUMO, MBP)

• Protein aggregation:

  • Solution: Screen multiple detergents (DDM, LMNG, CHAPS)

  • Solution: Add stabilizing agents (glycerol, specific lipids)

  • Solution: Express truncated constructs removing flexible regions

• Functional assays:

  • Solution: Develop binding assays with predicted partners

  • Solution: Assess membrane incorporation using fluorescence

  • Solution: Monitor cellular effects in overexpression systems

• Purification difficulties:

  • Solution: Implement on-column detergent exchange

  • Solution: Use size exclusion chromatography to remove aggregates

  • Solution: Test multiple buffer conditions via thermostability assays

These approaches build on established protocols for other membrane proteins while addressing the specific challenges of UPF0766 .

How can researchers validate antibodies for detecting endogenous UPF0766 protein?

A comprehensive antibody validation strategy includes:

• Expression controls:

  • Test antibody against recombinant UPF0766 protein

  • Compare with lysates from overexpression systems

  • Include knockout/knockdown samples as negative controls

• Specificity tests:

  • Perform peptide competition assays

  • Test cross-reactivity with related proteins

  • Compare multiple antibodies targeting different epitopes

• Application-specific validation:

  • For Western blotting: Confirm single band at expected MW

  • For immunoprecipitation: Verify pull-down efficiency with mass spectrometry

  • For immunohistochemistry: Compare with mRNA expression patterns

• Documentation requirements:

  • Record complete antibody information (catalog #, lot #, dilution)

  • Include all controls in publications

  • Report antibody validation methods

This systematic approach ensures reliable detection of endogenous UPF0766 protein .

What genomic and transcriptomic approaches might help elucidate the function of UPF0766 protein?

Advanced genomic and transcriptomic approaches for functional characterization:

• CRISPR-Cas9 screening:

  • Perform genome-wide knockout screen in relevant cell types

  • Look for synthetic lethality with UPF0766 knockout

  • Identify genetic interactions through combinatorial approaches

• Single-cell RNA-seq:

  • Compare expression patterns across tissues and cell types

  • Identify co-expressed gene networks

  • Track expression changes during development or disease progression

• Ribosome profiling:

  • Assess translational regulation of UPF0766

  • Identify potential upstream open reading frames

  • Determine translation efficiency under different conditions

• ChIP-seq and ATAC-seq:

  • Map transcription factor binding sites in the promoter region

  • Assess chromatin accessibility changes in different cell states

  • Identify epigenetic regulation mechanisms

These approaches can reveal functional associations and regulatory mechanisms controlling UPF0766 expression .

How might structural biology techniques be applied to understand UPF0766 protein function?

Structural biology approaches tailored for UPF0766:

• Cryo-electron microscopy (cryo-EM):

  • Particularly suitable if UPF0766 forms larger complexes

  • May require expression in nanodisc systems to maintain native environment

  • Resolution potential: 2.5-4Å for membrane proteins

• Nuclear magnetic resonance (NMR):

  • Well-suited for the small size of UPF0766 (88 amino acids)

  • Requires isotope labeling (15N, 13C)

  • Can provide dynamic information about protein motion

• X-ray crystallography:

  • Challenging for membrane proteins but possible with:

    • Lipidic cubic phase crystallization

    • Fusion to crystallization chaperones (T4 lysozyme)

    • Antibody fragment co-crystallization

• Integrative structural approaches:

  • Combine computational modeling with experimental constraints

  • Use crosslinking mass spectrometry to establish distance restraints

  • Validate models through mutagenesis of predicted functional sites