Recombinant Human Uncharacterized protein C16orf92 (C16orf92)

What is C16orf92 and where is it located in the human genome?

C16orf92 (chromosome 16 open reading frame 92) is a protein-coding gene located on chromosome 16 of the human genome. This gene encodes an uncharacterized protein with limited functional annotation in current research literature. The gene is identified by Entrez Gene ID 146378 and is categorized as a protein-coding sequence in the human genome . The official full name, "chromosome 16 open reading frame 92," reflects its status as a gene with unknown function that was identified through genomic sequencing efforts. Understanding the precise chromosomal location is essential for contextualizing potential functional relationships with nearby genes and regulatory elements.

What expression systems are most effective for producing recombinant C16orf92 protein?

Multiple expression systems have been employed for recombinant C16orf92 production, with bacterial and mammalian systems showing different advantages depending on research goals. For structural studies requiring high protein yields, E. coli-based expression systems using vectors like pET or pGEX are often preferred, though protein solubility may be challenging. For functional studies requiring proper folding and post-translational modifications, mammalian expression systems (HEK293 or CHO cells) using vectors with strong promoters like CMV may provide better results.

For researchers encountering solubility issues with C16orf92 expression, fusion partners such as MBP (maltose-binding protein) have proven effective in improving solubility. This approach was successfully demonstrated with other challenging proteins like components of the CIRTS-1 system, where direct overexpression led to insoluble protein until researchers resolved this by adding an N-terminal MBP tag . The purification strategy should include appropriate buffer optimization and may require detergent screening if C16orf92 exhibits membrane association properties.

What tools are available for CRISPR-mediated studies of C16orf92?

Researchers interested in CRISPR-based manipulation of C16orf92 have several commercially available tools at their disposal. Ready-to-use lentiviral particles containing gRNA targeting C16orf92 along with Cas9 nuclease are available with titers exceeding 1×10^7 IU/mL . These systems typically utilize a lentiviral vector backbone (e.g., pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro) with dual promoters: U6 driving sgRNA expression and SFFV promoting Cas9 expression .

When designing a CRISPR experiment targeting C16orf92, researchers should consider:

• Selection strategy: Available constructs contain puromycin resistance markers with ampicillin resistance for bacterial selection during cloning

• Expression stability: Both stable and transient expression options are supported by current vector systems

• Cell type compatibility: The SFFV promoter used in many constructs works effectively in most cell types except embryonic stem cells and induced pluripotent stem cells

• Quality control: Restriction enzyme digestion and sequencing verification using primers like the U6 Forward Primer (5'-TACGTCCAAGGTCGGGCAGGAAGA-3') are recommended before experimental use

Proper storage at -80°C is essential for maintaining viral particle viability, with typical shelf lives around 12 months under proper storage conditions .

Is C16orf92 potentially implicated in the phenotypic spectrum of 16p11.2 chromosomal rearrangements?

The relationship between C16orf92 and the well-documented 16p11.2 chromosomal region disorders presents an intriguing research direction. The 16p11.2 region harbors multiple segmental duplication loci that mediate recurrent CNVs (Copy Number Variants) through non-allelic homologous recombination (NAHR) . These CNVs are strongly associated with autism spectrum disorder (ASD), schizophrenia, intellectual disability, abnormal body weight, head circumference abnormalities, and various dysmorphic features .

Two key regions in 16p11.2 are frequently affected by CNVs:

• A distal interval of ~220 Kb between Breakpoints 2 and 3 (BP2–BP3)

• A proximal interval of ~593 Kb between Breakpoints 4 and 5 (BP4–BP5)

Researchers investigating C16orf92 should consider whether this gene falls within either of these critical regions, as its involvement could potentially contribute to the observed phenotypic spectrum. The variable expressivity and reduced penetrance observed in 16p11.2 rearrangement carriers might also apply to C16orf92 variants if the gene plays a role in neurodevelopment or metabolism .

Methodological approaches to investigate this question include:

• Precise mapping of C16orf92 relative to the BP2-BP5 region

• Expression analysis in patients with 16p11.2 CNVs

• Functional studies in model systems with C16orf92 knockdown or overexpression

• Phenotypic comparison between C16orf92 mutation carriers and 16p11.2 CNV patients

What RNA-Seq analysis approaches are most informative for investigating C16orf92 expression patterns?

RNA-Seq provides valuable insights into C16orf92 expression patterns across tissues, developmental stages, and disease states. When designing RNA-Seq experiments targeting C16orf92, researchers should consider both technical and analytical factors for optimal results.

For data collection, it's essential to obtain raw count data rather than only normalized values. Many public repositories like NCBI's Gene Expression Omnibus (GEO) contain RNA-Seq experiments with various data formats, but those providing raw counts are most valuable for differential expression analysis . Researchers should avoid datasets that only provide normalized values like FPKM or TPM, as these limit statistical analysis options .

For data analysis, consider this workflow:

• Quality Control: Assess read quality using tools like FastQC before mapping

• Read Alignment: Map reads to the reference genome using STAR or HISAT2

• Quantification: Count reads mapping to C16orf92 using HTSeq or featureCounts

• Normalization: Apply appropriate normalization methods (DESeq2, edgeR)

• Differential Expression Analysis: Compare C16orf92 expression across conditions

• Co-expression Network Analysis: Identify genes with similar expression patterns

• Functional Enrichment: Determine biological processes associated with co-expressed gene clusters

Since C16orf92 is uncharacterized, researchers should pay particular attention to co-expression networks, which may provide clues about its function through guilt-by-association approaches. Temporal expression data across developmental stages can be especially informative for genes with unknown functions.

How can researchers effectively design recombinant C16orf92 constructs for structure-function studies?

Designing optimal expression constructs is crucial for successful structure-function studies of uncharacterized proteins like C16orf92. Based on approaches used for other challenging proteins, researchers should consider a systematic construct design strategy.

When faced with expression or solubility challenges, fusion partners have proven effective. For example, in the development of CRISPR-Cas-inspired RNA targeting systems, researchers encountered insoluble protein until they fused an N-terminal MBP tag to their construct . This approach significantly improved solubility while maintaining protein functionality.

For C16orf92 structure-function studies, consider these methodological approaches:

• Domain Prediction and Truncation Series:

  • Use bioinformatics tools (InterPro, SMART, Pfam) to predict potential functional domains

  • Design multiple constructs with systematic N- and C-terminal truncations

  • Test expression and solubility of each construct in parallel

• Fusion Partner Screening:

  • Test multiple fusion tags: MBP, GST, SUMO, TRX, NusA

  • Position tags at both N- and C-termini to determine optimal configuration

  • Include precision protease cleavage sites (TEV or PreScission) for tag removal

• Codon Optimization:

  • Optimize codons for the expression system of choice

  • Remove rare codons that might cause translation pausing

  • Eliminate potential RNA secondary structures in the coding sequence

This systematic approach maximizes the likelihood of obtaining properly folded protein suitable for both structural and functional analyses.

What are the most effective ways to design and validate CRISPR-Cas9 experiments targeting C16orf92?

CRISPR-Cas9 genome editing provides powerful tools for investigating C16orf92 function, but requires careful experimental design and validation. For researchers using commercially available C16orf92 CRISPR tools like lentiviral particles with gRNA and Cas9 , or designing their own systems, the following methodological considerations are crucial:

• gRNA Design and Selection:

  • Design 3-4 gRNAs targeting different exons of C16orf92

  • Prioritize early exons to maximize disruption likelihood

  • Use algorithms that predict off-target effects and gRNA efficiency

  • Consider functional domains (if predicted) when selecting target sites

• Delivery Method Selection:

  • Lentiviral delivery: Suitable for difficult-to-transfect cells, provides stable integration

  • Ribonucleoprotein (RNP) complex: Reduces off-target effects due to transient Cas9 presence

  • Plasmid transfection: Economical but varies in efficiency across cell types

• Validation Strategy:

  • Genomic PCR and sequencing across the cut site to verify indel formation

  • T7 Endonuclease I assay for rapid screening of editing efficiency

  • Western blot to confirm protein depletion (if antibodies are available)

  • RT-qPCR to assess transcript level changes and potential nonsense-mediated decay

• Phenotypic Analysis:

  • Compare multiple independently derived knockout clones

  • Include rescue experiments to confirm phenotype specificity

  • Analyze cell proliferation, morphology, and relevant functional assays

  • Consider RNA-Seq to identify affected pathways

When working with lentiviral CRISPR systems targeting C16orf92, researchers should verify integration and expression using the recommended quality control measures, including restriction enzyme digestion and sequencing with appropriate primers like the U6 Forward Primer (5'-TACGTCCAAGGTCGGGCAGGAAGA-3') .

How can researchers investigate potential protein-protein interactions of C16orf92?

Investigating the interactome of uncharacterized proteins like C16orf92 provides crucial functional insights. Several complementary approaches can be employed:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged C16orf92 (FLAG, HA, or BioID) in relevant cell types

  • Perform pull-down under native conditions to maintain interactions

  • Identify copurifying proteins by mass spectrometry

  • Verify key interactions through reciprocal pull-downs

  • Filter against common contaminant databases

• Proximity-Based Labeling:

  • Fuse C16orf92 to BioID2 or TurboID biotin ligase

  • Express fusion protein in cells and provide biotin

  • Purify biotinylated proteins within proximity

  • Identify proteins by mass spectrometry

  • This approach captures both stable and transient interactions

• Yeast Two-Hybrid Screening:

  • Use C16orf92 as bait against human cDNA libraries

  • Screen for positive interactions through reporter gene activation

  • Verify positive hits through alternative methods

  • Consider membrane split-ubiquitin system if C16orf92 shows membrane association

• Co-Immunoprecipitation of Predicted Interactors:

  • Use bioinformatics to predict potential interactors based on:

    • Co-expression patterns in RNA-Seq data

    • Structural similarity to proteins with known interactions

    • Presence in the same subcellular compartment

  • Test these predictions through targeted co-immunoprecipitation

When analyzing interaction data, researchers should be cautious about false positives and prioritize interactions replicated across methods or supported by functional evidence. Understanding C16orf92's protein interaction network will provide valuable clues about its biological role and potential disease associations.

What are the future research directions for understanding C16orf92 function?

The uncharacterized nature of C16orf92 presents numerous opportunities for groundbreaking research. Future investigations should pursue multidisciplinary approaches to elucidate its function:

• Comprehensive Expression Profiling: Determine C16orf92 expression patterns across tissues, developmental stages, and disease states using RNA-Seq and proteomics approaches. Special attention should be paid to expression in the central nervous system given the association of the 16p11.2 region with neurodevelopmental disorders .

• Evolutionary Analysis: Examine conservation patterns across species to identify functionally important regions and potential homologs with known functions. This comparative genomics approach may reveal evolutionary constraints suggesting functional importance.

• Spatial Organization: Determine C16orf92's precise chromosomal location relative to the 16p11.2 regions associated with neuropsychiatric disorders. If located within or near these regions, C16orf92 may contribute to the phenotypic spectrum observed in patients with copy number variations .

• Functional Genomics: Apply systematic CRISPR-Cas9 knockout/knockdown strategies in relevant cell types and model organisms to observe resulting phenotypes. This approach, facilitated by available CRISPR resources targeting C16orf92 , can reveal cellular functions and pathways affected by C16orf92 absence.

• Protein Structure Determination: Pursue structural biology approaches (X-ray crystallography, cryo-EM, NMR) on purified recombinant C16orf92 to identify structural features that might suggest function. Fusion tags like MBP have proven useful for improving protein solubility in similar challenging cases .

• Integration with 16p11.2 Research: Explore potential contributions of C16orf92 to the "mirror phenotypes" observed in 16p11.2 CNV carriers, where deletions and duplications lead to opposite effects on head circumference and body weight .