Recombinant Macaca fascicularis Transmembrane protein C15orf27 homolog (QccE-15063)

What is the Recombinant Macaca fascicularis Transmembrane protein C15orf27 homolog?

The Recombinant Macaca fascicularis Transmembrane protein C15orf27 homolog (QccE-15063) is a recombinant protein derived from crab-eating macaques (Cynomolgus monkeys). It is a transmembrane protein with 417 amino acids in its full-length form. This protein is encoded by the QccE-15063 gene and corresponds to UniProt accession number Q9N0B5. The protein has multiple transmembrane domains and functions as a structural component of cellular membranes in this non-human primate model system .

How does the Macaca fascicularis C15orf27 homolog compare to the human version (TMEM266)?

The Macaca fascicularis C15orf27 homolog shares significant sequence similarity with the human C15orf27 (also known as TMEM266). The human protein consists of 581 amino acids and includes similar transmembrane domains. Comparative analysis reveals conserved functional domains between the species, although the human version contains additional regulatory regions. Human TMEM266 is expressed in HEK293T cells for recombinant production with a predicted molecular weight of 58.3 kDa when tagged with C-Myc/DDK . Researchers should note these differences when designing cross-species studies or when using the macaque protein as a model for human protein function .

What expression systems are suitable for producing QccE-15063?

Several expression systems can be utilized for the production of QccE-15063, each with advantages depending on experimental requirements:

The selection should be based on downstream application requirements and whether post-translational modifications are crucial for functional studies .

What purification strategies optimize yield and functionality of QccE-15063?

Optimal purification of QccE-15063 typically involves a multi-step approach:

• Affinity chromatography using fusion tags (His, FLAG, MBP, or GST) as the primary capture step

• Tag removal using specific proteases if tag-free protein is required

• Secondary purification using ion exchange chromatography to remove contaminants

• Size exclusion chromatography for final polishing and buffer exchange

• Optional endotoxin removal for cell-based assays

For membrane proteins like C15orf27 homolog, detergent selection during extraction and purification is critical. Mild detergents such as DDM (n-Dodecyl β-D-maltoside) or LMNG (Lauryl Maltose Neopentyl Glycol) often preserve protein structure while efficiently solubilizing the transmembrane domains .

How can researchers optimize storage conditions to maintain QccE-15063 stability?

The optimal storage conditions for maintaining QccE-15063 stability are:

• Short-term storage (1-2 weeks): 4°C in Tris-based buffer with appropriate detergent

• Long-term storage: -20°C or preferably -80°C in Tris-based buffer containing 50% glycerol

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• For lyophilized preparations, store at -20°C with desiccant

Researchers should validate protein activity after storage using functional assays specific to transmembrane proteins. Storage buffer optimization might be necessary depending on downstream applications, particularly for structural studies requiring high protein homogeneity .

How can QccE-15063 be utilized in transgenic iPSC studies?

QccE-15063 can be strategically employed in induced pluripotent stem cell (iPSC) studies to investigate gene regulation and disease mechanisms. Implementation involves:

• Establishing baseline expression profiles of endogenous C15orf27 homologs in iPSCs using RNA-seq

• Generating transgenic iPSC lines expressing tagged QccE-15063 via lentiviral transduction or CRISPR knock-in approaches

• Performing eQTL (expression Quantitative Trait Loci) mapping to identify genetic variants that affect QccE-15063 expression

• Differentiating iPSCs into relevant cell types to study developmental regulation of the protein

• Combining with population-scale transcriptomics to identify rare regulatory variants affecting QccE-15063 function

This approach allows researchers to investigate the role of C15orf27 in pluripotency maintenance and cellular differentiation pathways. The i2QTL consortium has established methodologies for such studies using large-scale resources of iPSCs with matched genotype and RNA-seq data .

What immunodetection methods are most effective for QccE-15063?

Effective immunodetection of QccE-15063 requires consideration of its transmembrane nature and expression levels:

For tag-based detection (His, FLAG, Myc), commercial antibodies provide reliable results. For direct detection, custom antibodies against specific domains of QccE-15063 may be required, focusing on hydrophilic regions for better antigenicity .

How can researchers investigate protein-protein interactions involving QccE-15063?

To study protein-protein interactions involving this transmembrane protein:

• Co-immunoprecipitation (Co-IP) using tagged QccE-15063 as bait, followed by mass spectrometry to identify interacting partners

• Proximity labeling techniques such as BioID or APEX2 fused to QccE-15063 to identify proximal proteins in living cells

• Membrane yeast two-hybrid (MYTH) system specifically designed for membrane protein interactions

• Split-GFP complementation assays to visualize interactions in living cells

• Surface plasmon resonance or microscale thermophoresis for quantifying binding kinetics with purified proteins

When designing these experiments, researchers should consider the orientation of QccE-15063 in the membrane and ensure that fusion tags do not interfere with potential interaction sites. Crosslinking approaches may help capture transient interactions that are often critical for membrane protein function .

How can QccE-15063 be used to investigate trans-eQTL effects in non-human primate models?

Utilizing QccE-15063 for trans-eQTL studies in non-human primates involves sophisticated experimental and analytical approaches:

• Establish baseline expression profiles across multiple tissues in Macaca fascicularis cohorts

• Perform genome-wide genotyping and RNA-sequencing on these cohorts (minimum N>100 individuals)

• Conduct trans-eQTL mapping focusing on variants >2.5 Mb from the QccE-15063 gene body

• Apply stepwise regression to identify independent genetic effects influencing QccE-15063 expression

• Integrate findings with human datasets to identify conserved regulatory networks

This approach can reveal distant genetic elements that regulate QccE-15063 expression across the genome. Previous studies have identified trans-acting variants associated with gene expression in pluripotent cells by testing associations between protein-coding genes and targeted sets of variants, including GWAS variants. With sufficient statistical power, researchers can detect regulatory relationships that may be relevant to understanding complex phenotypes in both macaques and humans .

What structural biology approaches are suitable for characterizing QccE-15063 transmembrane domains?

For structural characterization of QccE-15063 transmembrane domains:

Starting with smaller domains or using fusion partners like BRIL (thermostabilized apocytochrome b562) may facilitate structural studies. For complete structural characterization, a combination of these approaches is often necessary to overcome limitations inherent to membrane protein structural biology .

How does QccE-15063 contribute to understanding evolutionary conservation of transmembrane signaling pathways?

QccE-15063 serves as a valuable model for evolutionary analysis of transmembrane signaling pathways:

• Perform phylogenetic analysis comparing C15orf27 homologs across primates and other mammals

• Identify conserved domains that may indicate functional importance across evolutionary time

• Analyze selection pressures on different protein domains using dN/dS ratios

• Compare tissue-specific expression patterns across species using transcriptomic data

• Investigate species-specific post-translational modifications that may alter protein function

Comparative analysis between macaque QccE-15063 and human TMEM266 reveals evolutionary constraints on transmembrane topology and functional domains. This comparative approach can highlight critical regions for membrane localization, protein-protein interactions, and signaling functions that have been maintained throughout primate evolution. Additionally, species-specific differences may provide insights into adaptive changes in membrane protein biology related to environmental or physiological adaptations .

What are common challenges in functional assays using QccE-15063 and how can they be addressed?

Researchers frequently encounter several challenges when conducting functional assays with QccE-15063:

When designing functional assays, researchers should first establish clear readouts based on predicted protein function. For transmembrane proteins like QccE-15063, assays may include membrane potential measurements, calcium flux assays, or protein translocation studies depending on the hypothesized function .

How should researchers approach experimental design when comparing human and macaque C15orf27 homologs?

When designing comparative studies between human TMEM266 and macaque QccE-15063:

• Perform sequence alignment to identify conserved and divergent regions to guide experimental design

• Express both proteins in identical cellular contexts to control for system-specific effects

• Design chimeric constructs swapping domains between species to identify functionally important regions

• Use species-matched interaction partners when studying protein-protein interactions

• Control for differences in post-translational modifications that may affect function

What quality control parameters should be monitored when working with recombinant QccE-15063?

Critical quality control parameters for recombinant QccE-15063 include:

• Purity assessment via SDS-PAGE and Western blot (target >85-95% purity)

• Protein concentration determination using multiple methods (Bradford/BCA/A280)

• Endotoxin testing for preparations used in cell-based assays (<0.1 EU/μg protein)

• Functional validation specific to anticipated protein activity

• Stability assessment under storage and experimental conditions

• Batch-to-batch consistency verification

For advanced applications, additional quality control measures may include mass spectrometry to confirm protein identity and intact mass, circular dichroism to verify secondary structure, and dynamic light scattering to assess homogeneity and aggregation state. Establishing these quality control metrics at the beginning of a research project ensures data reliability and reproducibility throughout the study .

How might QccE-15063 be utilized in comparative studies of neurodegenerative diseases across primate models?

QccE-15063 presents significant opportunities for investigating neurodegenerative disease mechanisms across primate models:

• Generate transgenic macaque models expressing disease-associated variants of QccE-15063

• Perform comparative proteomic analyses of membrane protein complexes in healthy versus diseased tissues

• Investigate the role of QccE-15063 in maintaining membrane integrity under pathological conditions

• Utilize iPSC-derived neuronal cultures from both human and macaque sources expressing tagged versions of C15orf27 homologs

• Develop cross-species drug screening platforms targeting conserved domains of C15orf27 homologs

This comparative approach leverages the evolutionary proximity of macaques to humans while providing experimental advantages of non-human primate models. By integrating findings from population-scale transcriptomics with targeted functional studies of QccE-15063, researchers can identify causal relationships between genetic variants, protein function, and disease phenotypes across species .

What emerging technologies will advance structural and functional studies of QccE-15063?

Several emerging technologies show promise for advancing QccE-15063 research:

These technologies will enable previously unattainable insights into the structure-function relationships of QccE-15063, particularly regarding its transmembrane topology, dynamic conformational changes, and integration into cellular signaling networks .