Recombinant Pig UPF0694 transmembrane protein C14orf109 homolog

What is UPF0694 transmembrane protein C14orf109 homolog and what are its alternative names?

UPF0694 transmembrane protein C14orf109 homolog is a 163-amino acid transmembrane protein encoded by the TMEM251 gene in Sus scrofa (pig). The protein is also commonly referred to as Transmembrane protein 251 (TMEM251) in scientific literature. This nomenclature reflects its classification as part of the uncharacterized protein family UPF0694, with homology to the human C14orf109 (chromosome 14 open reading frame 109) protein . The UniProt ID for this protein is Q6QA74, which serves as a unique identifier in protein databases and is essential for cross-referencing in research publications .

What is the molecular weight and predicted structure of this protein?

The TMEM251 protein has a reported molecular weight of approximately 19/15 kDa as determined by SDS-PAGE analysis . This dual molecular weight observation may represent different post-translational modifications or proteolytic processing. As a transmembrane protein, TMEM251 contains hydrophobic regions that integrate into cellular membranes, which is reflected in its amino acid composition showing stretches of hydrophobic residues. The protein sequence analysis suggests it contains multiple transmembrane domains, consistent with its classification as a transmembrane protein .

What expression systems are optimal for Recombinant Pig UPF0694 protein production?

The most documented and efficient expression system for Recombinant Pig UPF0694 transmembrane protein C14orf109 homolog is Escherichia coli . When expressing this protein, the following key parameters should be considered:

While E. coli is the predominant system, researchers working with membrane proteins may also consider alternative expression systems such as those based on Semliki Forest virus (SFV) vectors for mammalian expression, which have shown success with other challenging transmembrane proteins .

What are the optimal storage and reconstitution conditions for purified recombinant protein?

For optimal stability and activity of Recombinant Pig UPF0694 transmembrane protein C14orf109 homolog, the following storage and reconstitution protocols are recommended:

• Storage form: The protein is typically supplied as a lyophilized powder, which provides greater stability during shipping and long-term storage .

• Reconstitution protocol:

  • Centrifuge the vial briefly before opening to collect contents at the bottom

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

  • Add glycerol to a final concentration of 5-50% (recommended: 50%) to prevent freeze-thaw damage

• Storage conditions:

  • Store reconstituted protein in aliquots at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles

  • Working aliquots may be stored at 4°C for up to one week

• Buffer composition: The protein is typically provided in a Tris/PBS-based buffer with 6% Trehalose, pH 8.0, which helps maintain protein stability .

What antibodies are available for detection of Pig UPF0694/TMEM251 protein?

Several antibodies are available for experimental detection of TMEM251/UPF0694 transmembrane protein. Based on the search results, researchers have access to:

• Goat polyclonal antibody (Santa Cruz Biotechnology, sc-242031) raised against a peptide mapping within an internal region of C14orf109 of human origin. This antibody is reported to have cross-reactivity with the porcine homolog .

• Rabbit polyclonal antibody targeting TMEM251 in human samples, which has been validated in multiple experimental systems with supporting image data .

These antibodies can be used in various applications including:

• Western Blotting (recommended starting dilution 1:200, range 1:100-1:1000)

• Immunofluorescence (recommended starting dilution 1:50, range 1:50-1:500)

• Solid-phase ELISA (recommended starting dilution 1:30, range 1:30-1:3000)

What methods can be used to validate successful expression of recombinant UPF0694/TMEM251?

To validate the successful expression and purification of Recombinant Pig UPF0694 transmembrane protein, researchers should implement a multi-method approach:

• SDS-PAGE analysis: The purified protein should appear at approximately 19/15 kDa, with purity greater than 90% .

• Western blot detection: Using anti-His antibodies for the recombinant His-tagged version or specific antibodies against TMEM251 .

• Mass spectrometry: For precise molecular weight determination and verification of the amino acid sequence.

• Functional assays: While specific functions of this protein remain to be fully characterized, preliminary assessments of membrane integration may be performed using fractionation techniques.

• Protein concentration determination: Using standard methods such as Bradford or BCA assays, with adjustments for membrane protein characteristics.

How does the chromosomal location of TMEM251 relate to its function?

The TMEM251 gene in humans maps to chromosome 14q32.12, while the mouse homolog maps to chromosome 12 E . Chromosome 14 contains approximately 700 genes and 106 million base pairs, comprising about 3.5% of human cellular DNA. This chromosome encodes several important genes including presenilin 1 (PSEN1), which is associated with Alzheimer's disease, and SERPINA1, which when defective leads to α1-antitrypsin deficiency characterized by severe lung complications and liver dysfunction .

The location of TMEM251 on chromosome 14 places it in genomic proximity to genes involved in important cellular processes, which may provide context for understanding its function. While direct functional associations based on chromosomal location remain speculative, this information provides a starting point for investigating potential roles in cellular processes affected in diseases associated with this chromosomal region.

What are the challenges in structural characterization of UPF0694/TMEM251 protein?

Structural characterization of transmembrane proteins like UPF0694/TMEM251 presents several technical challenges:

• Protein solubility: Membrane proteins are inherently hydrophobic and difficult to maintain in solution without appropriate detergents or lipid environments.

• Crystallization difficulties: The presence of multiple transmembrane domains makes crystallization for X-ray crystallography challenging.

• Purification complexity: Obtaining sufficient quantities of pure, correctly folded protein often requires optimization of expression conditions and detergent screening.

• Functional reconstitution: Assessing proper folding and function may require reconstitution into artificial membrane systems.

To address these challenges, researchers should consider:

• Screening multiple detergents for protein extraction and stability

• Using techniques such as circular dichroism to assess secondary structure content

• Employing cryo-electron microscopy as an alternative to X-ray crystallography

• Developing functional assays that can verify proper protein folding and activity

What genetic manipulation strategies can be used to study UPF0694/TMEM251 function?

For genetic studies of UPF0694/TMEM251 function, several tools are available to researchers:

• RNA interference: Commercial siRNA products are available for targeting C14orf109 in human cells (sc-106903) and the mouse homolog D230037D09Rik (sc-142803) .

• shRNA plasmids: For stable knockdown, shRNA plasmids are available for both human C14orf109 (sc-106903-SH) and mouse D230037D09Rik (sc-142803-SH) .

• Lentiviral particles: For difficult-to-transfect cells, lentiviral particles carrying shRNA are available for human C14orf109 (sc-106903-V) and mouse D230037D09Rik (sc-142803-V) .

• CRISPR/Cas9 genome editing: Custom gRNAs can be designed to target the TMEM251 gene for knockout studies or to introduce specific mutations.

These genetic tools allow researchers to modulate TMEM251 expression levels and analyze resulting phenotypes to infer protein function in various cellular processes.

How conserved is UPF0694/TMEM251 across species?

The UPF0694/TMEM251 protein shows conservation across multiple species, suggesting functional importance in cellular processes. The available antibodies for this protein demonstrate cross-reactivity across several species:

The goat polyclonal antibody (sc-242031) is recommended for detection of C14orf109 in multiple species including human, mouse, rat, equine, canine, bovine, porcine, and avian samples . This broad cross-reactivity suggests significant sequence conservation across vertebrates.

The mouse homolog, identified as D230037D09Rik, maps to chromosome 12 E, showing synteny with the human chromosomal location . This conservation of genomic organization provides additional evidence for evolutionary preservation of this gene.

Detailed sequence alignment analysis would be necessary to determine specific regions of high conservation, which might indicate functional domains or interaction surfaces critical to the protein's role in cellular processes.