Recombinant Bovine Transmembrane protein C14orf180 homolog

What is Recombinant Bovine Transmembrane protein C14orf180 homolog?

Recombinant Bovine Transmembrane protein C14orf180 homolog (UniProt ID: Q29RM6) is a full-length protein (159 amino acids) also known as Nutritionally-regulated adipose and cardiac-enriched protein homolog (NRAC). The protein can be produced with an N-terminal His tag in E. coli expression systems. The complete amino acid sequence is: MKTAVHALSPDSRPETQHQTRKNEEAAPGSPTPRAGREGRKGPASILRRSPQERCGRGDEPRRTTRHVRFREPLEVAVHYIACREPTTAVQAPSRPRPRGGSLLLRLTACILLALALGMCCGQAGPMARALEDFRARLLAALLRLPLAALDCWRCLLQL .

What expression systems are suitable for producing this protein?

While E. coli is commonly used for expressing this recombinant protein , researchers should consider alternative eukaryotic expression systems that preserve post-translational modifications, especially for functional studies. For transmembrane proteins, mammalian expression systems like the PEAKrapid CRL-2828 human kidney cells can be advantageous as they maintain proper protein folding and post-translational modifications essential for biological activity .

What are the recommended storage conditions for maintaining protein stability?

For optimal stability, store the protein at -20°C/-80°C upon receipt with aliquoting necessary for multiple use. Avoid repeated freeze-thaw cycles as they may compromise protein integrity. The protein is typically provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0. For reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol (final concentration) recommended for long-term storage .

How should researchers design experiments to characterize the functional properties of this protein?

When designing functional characterization experiments, consider the following methodological approach:

• Begin with expression analysis across relevant bovine tissues to establish baseline expression patterns

• Implement cell-based assays in appropriate bovine cell lines (e.g., adipose or cardiac cells)

• Design loss-of-function studies using CRISPR/Cas9-mediated gene editing

• Perform protein localization studies using immunofluorescence or tagged protein variants

• Conduct comparative analysis with human or murine orthologs to infer conserved functions

For gene knockout experiments in bovine systems, electroporation has proven effective for CRISPR/Cas9 delivery to bovine zygotes with high editing efficiency .

What purification strategies are most effective for this transmembrane protein?

Given the N-terminal His tag on the recombinant protein, immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins is the primary purification method. For optimal results:

• Lyse cells in buffers containing appropriate detergents to solubilize membrane proteins

• Include imidazole in wash buffers (10-20 mM) to reduce nonspecific binding

• Use a step gradient elution with increasing imidazole concentrations

• Consider size exclusion chromatography as a polishing step to achieve >90% purity

• Validate purification success via SDS-PAGE analysis

For transmembrane proteins, inclusion of mild detergents (e.g., DDM or CHAPS) throughout the purification process is crucial to maintain protein solubility and native conformation.

What analytical methods should be employed for quality control?

Comprehensive quality control should include:

• SDS-PAGE analysis to verify protein purity (>90% recommended)

• Western blotting with anti-His antibodies to confirm identity

• Mass spectrometry for accurate molecular weight determination and sequence verification

• Circular dichroism spectroscopy to assess secondary structure integrity

• Dynamic light scattering to evaluate protein homogeneity and aggregation state

How can RNA-seq and bioinformatics approaches be utilized to study C14orf180 homolog expression patterns?

RNA-seq analysis provides powerful insights into expression patterns across tissues and developmental stages. Researchers should:

• Implement weighted co-expression network analysis (WGCNA) to identify gene networks associated with C14orf180 homolog

• Apply single gene set enrichment analysis (GSEA) to reveal enriched biological pathways

• Compare expression patterns across different bovine tissues using standardized RNA-seq datasets such as GSE137943

• Validate key findings with independent datasets like GSE186481

• Integrate transcriptomic data with proteomics for comprehensive functional characterization

Implementation of robust preprocessing methods including RMA (robust multichip average) for background correction and normalization is essential for reliable analysis .

What approaches can elucidate the protein-protein interaction network of C14orf180 homolog?

To identify interaction partners and construct a functional protein network:

• Perform co-immunoprecipitation with anti-His antibodies followed by mass spectrometry identification

• Implement proximity-dependent biotinylation approaches (BioID or TurboID)

• Utilize yeast two-hybrid screening with the soluble domains

• Conduct cross-linking mass spectrometry to identify interaction interfaces

• Validate key interactions through co-localization studies and functional assays

How can CRISPR/Cas9 gene editing be optimized for studying C14orf180 homolog function in bovine systems?

For effective CRISPR/Cas9 editing in bovine systems:

• Design multiple guide RNAs targeting conserved exons of the C14orf180 gene

• Optimize electroporation conditions for bovine cells (parameters from successful bovine zygote studies: voltage 30 V/mm, 3 ms pulse, 6 pulses at 0.1 s interval)

• Validate knockout efficiency through PCR amplification and Sanger sequencing

• Analyze indel patterns using bioinformatics tools such as ICE CRISPR Analysis Tool

• Calculate editing efficiency based on the proportion of cells with insertions/deletions

What strategies can overcome expression and solubility issues with this transmembrane protein?

Transmembrane proteins often present expression and solubility challenges. Implement these strategies:

• Optimize expression conditions (temperature reduction to 18-25°C, lowered inducer concentration)

• Test different E. coli strains designed for membrane protein expression (C41(DE3), C43(DE3))

• Consider fusion partners that enhance solubility (MBP, SUMO, Trx)

• Screen detergents systematically for extraction efficiency (n-Dodecyl β-D-maltoside, CHAPS, digitonin)

• Explore alternative expression systems such as insect cells or mammalian cells for proper folding

How can researchers address potential endotoxin contamination from E. coli-expressed proteins?

Endotoxin contamination can significantly impact downstream applications, particularly in cell-based assays. To address this issue:

• Implement Triton X-114 phase separation during purification

• Utilize polymyxin B-based affinity chromatography

• Include endotoxin removal steps in the purification workflow

• Test final preparations using Limulus Amebocyte Lysate (LAL) assay

• Consider eukaryotic expression systems when endotoxin sensitivity is critical

What approaches can resolve issues with post-translational modifications in bacterial expression systems?

Bacterial expression systems lack the machinery for many eukaryotic post-translational modifications. To address this limitation:

• Identify essential modifications through bioinformatic prediction tools

• Express protein in eukaryotic systems that preserve these modifications

• Consider engineered bacterial strains with enhanced post-translational capability

• For glycosylation studies, implement mammalian expression systems similar to those used for bovine GM-CSF and IL-4

• Evaluate functional impact of modifications through comparative activity assays

How should researchers analyze differential expression data involving C14orf180 homolog?

For robust differential expression analysis:

• Normalize RNA-seq data using appropriate methods (RMA recommended)

• Calculate expression levels as mean ± SEM from at least three independent experiments

• Apply appropriate statistical tests (t-test for two-group comparisons)

• Use GAPDH expression as endogenous control for normalization

• Consider results statistically significant when P < 0.05 (* P < 0.05 and ** P < 0.01)

What bioinformatic resources can assist in functional annotation of C14orf180 homolog?

To develop comprehensive functional annotations:

• Utilize UniProt database (entry Q29RM6) for sequence information and known functions

• Implement InterPro and Pfam for domain prediction and family classification

• Apply Gene Ontology enrichment analysis to identify associated biological processes

• Perform phylogenetic analysis with orthologs from other species to infer conserved functions

• Integrate findings from GEO datasets (GSE137943, GSE186481) to establish tissue-specific expression patterns