Recombinant Ailuropoda melanoleuca UPF0767 protein C1orf212 homolog (PANDA_005386)

What is UPF0767 protein C1orf212 homolog (PANDA_005386) and what do we know about its structure?

UPF0767 protein C1orf212 homolog (PANDA_005386), also known as SMIM12 (Small integral membrane protein 12), is a transmembrane protein identified in the giant panda (Ailuropoda melanoleuca) with a UniProt ID of D2H617. The full-length protein consists of 92 amino acids with the sequence: MWPVLWTVVRTYAPYVTFPVAFVVGAVGYHLEWFIRGKDPQPVEEEKSISERREDRKLDE LLGKDHTQVVSLKDKLEFAPKAVLNRNRPEKN . Based on current knowledge, this protein belongs to a family of small integral membrane proteins, though its specific function in pandas remains under investigation. Structural analysis techniques including circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) would be recommended for elucidating secondary and tertiary structures.

Which expression systems are most effective for producing recombinant UPF0767 protein C1orf212 homolog?

Multiple expression systems have been utilized successfully for this recombinant protein, with each offering distinct advantages depending on research objectives. E. coli-based expression systems have demonstrated the highest yield and shortest turnaround times, making them ideal for structural studies requiring substantial protein quantities . For applications requiring post-translational modifications for proper folding or activity retention, insect cells with baculovirus or mammalian expression systems are recommended . When expressing this protein, N-terminal His-tagging has been successfully implemented to facilitate purification . The optimal expression system selection should be guided by the specific experimental requirements, considering factors such as protein folding needs, yield requirements, and downstream applications.

What are the recommended storage conditions for maintaining stability of the purified protein?

For optimal stability preservation, the recombinant protein should be stored at -20°C for short-term or -80°C for extended storage periods . Lyophilized preparations generally maintain stability for up to 12 months at these temperatures, while liquid formulations have a shorter shelf life of approximately 6 months . To prevent protein degradation through freeze-thaw cycles, it is advisable to add glycerol to a final concentration of 5-50% (with 50% being commonly used) and aliquot the protein solution prior to storage . For working solutions, store aliquots at 4°C for no longer than one week . Prior to reconstitution, briefly centrifuge vials to bring contents to the bottom and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

How can I optimize purification protocols for UPF0767 protein C1orf212 homolog to maximize yield and purity?

Optimizing purification of this recombinant protein requires a multi-step approach focusing on both yield and purity. For His-tagged preparations, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin serves as an effective initial purification step . To achieve purity levels exceeding 85-90% as verified by SDS-PAGE, consider implementing the following protocol:

• Begin with cell lysis using sonication or mechanical disruption in a buffer containing 20-50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole

• Clarify lysate by centrifugation (15,000 × g for 30 minutes at 4°C)

• Apply supernatant to pre-equilibrated Ni-NTA column

• Wash with increasing imidazole concentrations (20-50 mM) to remove non-specific binding

• Elute protein with 250-300 mM imidazole

• For higher purity, implement size exclusion chromatography as a polishing step

• Dialyze against Tris/PBS-based buffer (pH 8.0) containing 6% trehalose

Adjust buffer conditions based on protein stability assessments and intended downstream applications.

What analytical methods are recommended for assessing the quality and integrity of the recombinant protein?

Several complementary analytical techniques should be employed to comprehensively evaluate protein quality:

For transmembrane proteins like PANDA_005386, additional detergent screening may be necessary to maintain proper folding during purification and analysis. Thermal shift assays can provide valuable insights into protein stability under various buffer conditions.

How does UPF0767 protein C1orf212 homolog relate to the immune system function in giant pandas?

While direct evidence linking UPF0767 protein C1orf212 homolog to immune function is limited, research on giant panda transcriptomics provides contextual insights. Transcriptome analysis of giant panda blood has identified 210 differentially expressed genes between young (2-6 years) and old (18-21 years) pandas, with 146 up-regulated and 64 down-regulated genes in older animals . The key immune-related hub genes identified include ISG15, STAT1, IRF7, and DDX58, which play crucial roles in responses to pathogen invasion . These findings reveal that innate immune response genes are predominantly up-regulated with age, potentially compensating for declining adaptive immune function .

To investigate potential relationships between UPF0767 protein C1orf212 homolog and immune function, researchers should consider:

• Analyzing co-expression networks between C1orf212 homolog and known immune regulators

• Examining expression profiles across different age groups and health conditions

• Conducting protein-protein interaction studies with identified immune hub genes

• Implementing functional assays to assess impact on immune signaling pathways

What methodologies should be employed to investigate potential functions of this poorly characterized protein?

Investigating an uncharacterized protein like UPF0767 protein C1orf212 homolog requires a systematic multi-omics approach:

• Comparative genomics analysis: Compare sequence homology and conserved domains across species to identify evolutionary relationships and potential functional motifs .

• Protein interaction studies:

  • Implement yeast two-hybrid screening

  • Conduct co-immunoprecipitation followed by mass spectrometry

  • Utilize proximity labeling approaches (BioID or APEX)

  • Perform genetic interaction screens

• Cellular localization determination:

  • Fluorescent protein tagging and confocal microscopy

  • Subcellular fractionation followed by Western blotting

  • Immunohistochemistry on giant panda tissues (if available)

• Gene expression modulation:

  • Generate knockout/knockdown models in relevant cell lines

  • Employ CRISPR-Cas9 for precise genetic manipulation

  • Conduct transcriptome analysis following gene modulation

• Functional assays based on predicted characteristics:

  • For transmembrane proteins, assess membrane integrity and transport functions

  • Investigate potential roles in signaling pathways

This comprehensive approach can generate hypotheses regarding protein function for further targeted investigation.

How can UPF0767 protein C1orf212 homolog inform our understanding of giant panda evolution and adaptation?

The giant panda (Ailuropoda melanoleuca) presents a fascinating case of evolutionary adaptation, having diverged from the ursid lineage prior to the radiation that led to modern bears . Molecular and genetic analyses have resolved the phylogenetic placement of giant pandas, showing they split from the bear lineage more recently than the lesser panda diverged from New World procyonids . This divergence was accompanied by significant chromosomal reorganization .

Studying UPF0767 protein C1orf212 homolog within this evolutionary context can provide insights into:

• Adaptive molecular evolution: Comparative analysis of this protein across carnivores can reveal selection pressures and adaptive mutations specific to the giant panda lineage.

• Dietary specialization: Given the giant panda's unique bamboo diet requiring specialized digestive adaptations , investigating whether this protein contributes to dietary metabolism or nutrient absorption pathways.

• Functional conservation: Determining whether protein structure and function are conserved among bears despite the giant panda's dietary specialization, or if divergence has occurred.

Methodological approaches should include positive selection analysis, protein structure prediction, and comparative expression studies across tissues involved in bamboo digestion and metabolism.

What role might this protein play in giant panda dietary adaptations to bamboo consumption?

Giant pandas have evolved specialized dietary adaptations for bamboo consumption, requiring a balanced intake of different bamboo parts (culm, leaves, and shoots) for optimal metabolic function . Research indicates pandas consuming only culm absorb higher amounts of calories and fiber but experience energy supply shortages (depressed tricarboxylic acid cycle activity) . Protein intake appears crucial for body mass increase, with shoot-consuming pandas showing higher growth rates .

To investigate potential roles of UPF0767 protein C1orf212 homolog in these dietary adaptations:

• Expression analysis: Compare protein expression levels across digestive tissues in pandas fed different bamboo parts

• Metabolic pathway integration: Determine if the protein participates in fiber digestion or energy metabolism pathways

• Comparative analysis: Assess functional differences between this protein in pandas versus non-bamboo-consuming relatives

• Nutrient sensing: Investigate potential roles in nutrient sensing or metabolic regulation pathways

Given the protein's transmembrane nature, it could potentially function in nutrient transport or signaling processes related to metabolic adaptation to bamboo consumption.

How can UPF0767 protein C1orf212 homolog be utilized in developing immunological biomarkers for monitoring giant panda health?

Building on the transcriptomic evidence showing age-related changes in immune gene expression in giant pandas , UPF0767 protein C1orf212 homolog could potentially serve as part of a biomarker panel for monitoring health and immunological status. A comprehensive approach would include:

• Longitudinal expression profiling: Monitor protein levels in blood samples from pandas of various ages and health statuses to establish baseline expression patterns and identify correlations with health parameters.

• Multi-biomarker panel development: Integrate with known immune hub genes (ISG15, STAT1, IRF7, DDX58) to create a comprehensive immunological assessment panel.

• Antibody development methodology:

  • Generate monoclonal antibodies against specific epitopes of UPF0767 protein C1orf212 homolog

  • Validate antibody specificity using Western blot against recombinant protein

  • Develop ELISA or other immunoassay platforms for quantitative detection

• Clinical validation process:

  • Correlate biomarker levels with comprehensive health assessments

  • Establish reference ranges across different age groups

  • Determine sensitivity and specificity for detecting disease states

  • Validate in multiple panda populations

This approach would require close collaboration between molecular biologists, veterinarians, and conservation biologists to translate laboratory findings into practical monitoring tools.

What are the challenges and solutions for structural determination of this transmembrane protein?

Structural determination of transmembrane proteins like UPF0767 protein C1orf212 homolog presents significant challenges requiring specialized methodologies:

For this 92-amino acid transmembrane protein, NMR spectroscopy may be particularly suitable given the relatively small size. Expression with isotope labeling (15N, 13C) in minimal media would be required for structural determination by NMR. Alternatively, computational approaches using AlphaFold2 or RoseTTAFold could provide initial structural models to guide experimental design.

What are the most promising future research directions for UPF0767 protein C1orf212 homolog?

Based on current understanding and technical capabilities, several high-priority research directions emerge:

• Comprehensive functional characterization: Employing CRISPR-Cas9 gene editing in relevant cell models to determine essential functions and pathway involvement.

• Integration with giant panda conservation research: Correlating protein expression with health parameters in captive and wild populations to develop monitoring tools.

• Structural biology advances: Determining three-dimensional structure to enable structure-based functional predictions and evolutionary analysis.

• Developmental biology perspectives: Investigating expression patterns throughout different life stages to identify potential roles in growth and development.

• Comparative immunology: Examining potential immunological functions in the context of age-related changes in giant panda immune systems .

Each of these directions requires interdisciplinary collaboration between molecular biologists, conservation scientists, structural biologists, and wildlife veterinarians to advance our understanding of this protein and its potential significance in giant panda biology.