Recombinant Bovine UPF0708 protein C6orf162 homolog

What is UPF0708 protein C6orf162 homolog and how is it classified?

UPF0708 protein C6orf162 homolog is a small integral membrane protein (SMIM8) located on chromosome 6. It belongs to the family of proteins with uncharacterized protein function (UPF) designation, indicating its biological role is not yet fully elucidated. The protein is characterized by an alpha-helical structure, which is the most common structural motif in transmembrane proteins . This classification is significant as alpha-helical proteins are considered suitable targets for therapeutics and diagnostics, similar to other cell surface membrane proteins .

What expression systems are typically used for recombinant production of UPF0708 protein C6orf162 homolog?

The primary expression system used for recombinant production of UPF0708 protein C6orf162 homolog is Escherichia coli (E. coli). This prokaryotic expression system has demonstrated successful expression of both mouse and bovine homologs of the protein . For the mouse homolog, the recombinant protein is typically fused to an N-terminal His tag to facilitate purification . Expression yields of similar recombinant proteins have been reported at approximately 34 mg/mL under optimized conditions . The recombinant protein is commonly stored as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

What is the amino acid sequence and structural characteristics of UPF0708 protein C6orf162 homolog?

Based on data from the mouse homolog, the full-length protein consists of 97 amino acids with the following sequence: MSSAPDPPTVKKEPLKEKNFENPGLRGAHTTTLFRAVNPELFIKPNKPVMAFGLVTLSLCVAYIGYLHATQENRKDLYEAIDSEGHRYMRRKTSKWD . Structural analysis through bioinformatics tools like ColabFold reveals that this protein is primarily composed of alpha-helices, which is typical for transmembrane proteins . Some portions of the protein sequence show similarity to the ESAT-6 family (UNIPROT accession number: P64094), which is notable for its potential roles in virulence and as diagnostic candidates .

What are the optimal conditions for reconstitution and storage of recombinant UPF0708 protein C6orf162 homolog?

For optimal reconstitution of lyophilized recombinant UPF0708 protein C6orf162 homolog, the following methodology is recommended:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is standard) to stabilize the protein for long-term storage

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep aliquots at -20°C/-80°C

Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity and activity .

How can antigenicity of UPF0708 protein C6orf162 homolog be evaluated for diagnostic applications?

Antigenicity evaluation follows a systematic approach similar to that used for other mycobacterial proteins:

• Initial computational prediction using software such as ANTIGENPro, with scores ≥0.5 considered antigenic

• Production of recombinant protein with appropriate tagging for purification

• Concentration optimization for in vivo testing, starting with ranges from 0.0004 to 0.04 μg per 100 μL for preliminary screening

• Testing higher concentrations (40-80 μg) based on initial response data

• In vivo evaluation in sensitized and non-sensitized animal models (e.g., guinea pigs)

• Measurement of intradermal skin reactions at specific timepoints (24h, 48h, and 72h), with 72h readings typically providing the most accurate results

• Comparison of induration response between test subjects and controls to determine diagnostic potential

What purification strategies are most effective for recombinant UPF0708 protein C6orf162 homolog?

Given the N-terminal His tag commonly used in recombinant expression of UPF0708 protein C6orf162 homolog, the following purification strategy is recommended:

• Cell lysis under native or denaturing conditions depending on protein solubility

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture His-tagged protein

• Washing with buffer containing low concentrations of imidazole to remove non-specifically bound proteins

• Elution with buffer containing high imidazole concentration

• Buffer exchange to remove imidazole using dialysis or size exclusion chromatography

• Quality control through SDS-PAGE to confirm purity (target >90% purity)

• Concentration determination using established protein quantification methods

• Lyophilization in stabilizing buffer containing 6% trehalose

How does UPF0708 protein C6orf162 homolog compare to traditional PPD antigens in bovine tuberculosis diagnostics?

Comparative analysis between UPF0708 protein C6orf162 homolog and purified protein derivative (PPD) antigens reveals several key differences:

• Specificity: UPF0708 protein C6orf162 homolog, like other specific recombinant proteins, may offer improved specificity compared to PPD antigens, which contain numerous cross-reactive components that can lead to false-positive results .

• Standardization: Recombinant proteins can be produced with consistent quality and composition, addressing a major limitation of PPD antigens which can vary between batches .

• Intradermal response kinetics: In sensitized animals, recombinant proteins like UPF0708 protein C6orf162 homolog may show different temporal patterns of skin reaction compared to PPD. While PPD reactions may be detectable at 24h, specific recombinant proteins often reach peak reaction at 72h post-injection .

• Differentiation capacity: In experimental models, specific recombinant proteins have demonstrated superior ability to differentiate between sensitized and non-sensitized animals compared to PPD, showing no perceptible reaction in control animals while maintaining robust response in sensitized subjects .

What bioinformatic approaches can be used to identify potential diagnostic applications of UPF0708 protein C6orf162 homolog?

A comprehensive bioinformatic pipeline for evaluating UPF0708 protein C6orf162 homolog as a diagnostic candidate includes:

• Pangenome analysis across multiple related species (e.g., Mycobacterium species) to identify unique genes/proteins

• Multiple sequence alignment with less than 60% identity threshold to identify species-specific protein variants

• Antigenicity prediction using specialized software (e.g., ANTIGENPro), with a threshold of ≥0.5 considered antigenic

• Structural prediction using tools like ColabFold to visualize protein architecture

• Identification of genomic islands associated with virulence and pathogenesis

• Evaluation of sequence similarity to known virulence factors or diagnostic targets (e.g., ESAT-6 family)

• Transmembrane topology prediction to assess membrane localization potential

• Epitope mapping to identify potential T-cell and B-cell recognition sites

This integrated approach has successfully identified novel antigens for bovine tuberculosis diagnosis, providing a framework for evaluation of UPF0708 protein C6orf162 homolog.

What concentration optimization strategies should be employed for intradermal testing of UPF0708 protein C6orf162 homolog?

Concentration optimization for intradermal testing follows a systematic approach based on established methodologies:

The 72-hour reading timepoint is critical as it provides the most accurate assessment of reaction, particularly for recombinant proteins. At this timepoint, sensitized animals show clear skin reactions while non-sensitized controls exhibit no perceptible reaction, enabling clear differentiation between groups . This time point aligns with official bovine tuberculosis diagnostic protocols established by regulatory agencies .

What are the primary technical challenges in producing functional recombinant UPF0708 protein C6orf162 homolog?

The production of functional recombinant UPF0708 protein C6orf162 homolog faces several technical challenges:

• Membrane protein expression: As a small integral membrane protein, expression can be complicated by protein folding issues, toxicity to host cells, and low yields.

• Structural integrity preservation: Maintaining the alpha-helical structure during purification is crucial for preserving functionality.

• Solubility limitations: Membrane proteins often have limited solubility in aqueous solutions, requiring optimization of buffer conditions.

• Scale-up consistency: Ensuring consistent protein quality and activity during scaled-up production for experimental applications.

• Species-specific variations: Differences between bovine, mouse, and human homologs may require specific optimization for each species variant.

• Post-translational modifications: If present in the native protein, these may not be properly reproduced in bacterial expression systems like E. coli .

How can researchers assess cross-reactivity concerns with UPF0708 protein C6orf162 homolog in diagnostic applications?

Cross-reactivity assessment for UPF0708 protein C6orf162 homolog requires a multi-faceted approach:

• Computational analysis: Compare protein sequences across multiple Mycobacterium species and related organisms to identify regions of high similarity.

• Sensitization studies: Test animals sensitized with different Mycobacterium species to evaluate specificity of skin test reactions.

• Combinatorial testing: Evaluate the recombinant protein alongside other antigens to determine if combined use improves diagnostic accuracy.

• Field validation: Test the protein in naturally infected and non-infected animals in diverse geographic regions to assess real-world specificity.

• Epitope mapping: Identify specific epitopes that may contribute to cross-reactivity and consider protein engineering to enhance specificity.

• Serum antibody cross-adsorption: Perform pre-adsorption of test sera with related antigens to assess specific versus cross-reactive antibody responses .

What are the potential applications of UPF0708 protein C6orf162 homolog beyond tuberculosis diagnostics?

Based on structural and functional characteristics of UPF0708 protein C6orf162 homolog, several potential applications beyond tuberculosis diagnostics can be explored:

• Target for antimicrobial development: As a membrane protein potentially involved in pathogenesis, it may serve as a novel drug target.

• Vaccine component: If sufficiently immunogenic, it could be evaluated as a component in subunit vaccines.

• Biomarker for disease progression: Expression levels might correlate with disease states or bacterial load.

• Research tool for membrane protein studies: As a model alpha-helical membrane protein, it could inform structural biology research.

• Comparative genomics model: The conservation and variation of this protein across species makes it valuable for evolutionary studies.

• Cell biology investigations: Understanding its natural function could provide insights into basic cellular processes .