Recombinant UPF0197 transmembrane protein Y57E12AM.1 (Y57E12AM.1)

What is UPF0197 transmembrane protein Y57E12AM.1 and what are its key properties?

UPF0197 transmembrane protein Y57E12AM.1 is a protein found in Caenorhabditis elegans with UniProt ID Q965T1. It is also known by several synonyms including tmem-258, Transmembrane protein 258, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258, and Oligosaccharyl transferase subunit TMEM258 . The full-length protein consists of 79 amino acids with the sequence: MDISKMNRYTAPVNFASLPLLTTFLCGVGLLLLATFTMIQVTSTKYNRNLLKELFIAATSSVFLGFGSVFLLLWVGIYV . As suggested by its name, it belongs to the UPF0197 family of transmembrane proteins, which are still being characterized in terms of their specific biological functions.

Which expression systems are most effective for producing recombinant Y57E12AM.1?

E. coli is the most commonly documented expression system for recombinant Y57E12AM.1 production . When expressing transmembrane proteins like Y57E12AM.1, optimized induction conditions are critical to maximize soluble protein yield while minimizing inclusion body formation. Based on established protocols for similar recombinant proteins, effective expression can be achieved by growing the culture to an OD600 of approximately 0.8 before inducing with 0.1 mM IPTG, followed by expression at lower temperatures (25°C) for 4 hours in a medium containing 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, and 1 g/L glucose . This approach has been shown to significantly improve the soluble expression of recombinant proteins in E. coli.

What are the recommended methods for purifying recombinant Y57E12AM.1?

For His-tagged recombinant Y57E12AM.1, immobilized metal affinity chromatography (IMAC) is the primary purification method . The purification protocol should be optimized to achieve greater than 90% purity as determined by SDS-PAGE . After initial purification, size exclusion chromatography can be employed to further enhance purity and remove protein aggregates. When working with transmembrane proteins like Y57E12AM.1, it's important to select appropriate detergents during purification to maintain protein stability and native conformation. The specific detergent choice would depend on downstream applications and the protein's biophysical properties.

What are the optimal storage conditions for maintaining Y57E12AM.1 stability?

Recombinant Y57E12AM.1 is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt . For long-term storage, the protein should be reconstituted and aliquoted to avoid repeated freeze-thaw cycles, which can significantly reduce protein activity and stability . The recommended storage buffer consists of a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . For extended storage periods, addition of glycerol to a final concentration of 5-50% is recommended, with 50% being the standard concentration used by many suppliers . Working aliquots may be stored at 4°C for up to one week .

What is the recommended reconstitution protocol for lyophilized Y57E12AM.1?

For optimal reconstitution of lyophilized Y57E12AM.1:

• Briefly centrifuge the vial before opening to ensure all content is collected 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% for long-term storage stability

• Prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted protein at -20°C/-80°C for long-term storage, or at 4°C for up to one week for working solutions

What experimental approaches can optimize soluble expression of Y57E12AM.1?

When designing experiments for Y57E12AM.1 expression, a factorial design approach can significantly improve yields of functional protein. Key variables to consider include:

• Induction timing (optimal OD600)

• Inducer concentration (IPTG concentration)

• Post-induction temperature

• Expression duration

• Media composition

• Antibiotic concentration

Research on similar recombinant proteins suggests that lower post-induction temperatures (25°C vs. 37°C) and moderate inducer concentrations (0.1 mM IPTG) often result in higher yields of correctly folded transmembrane proteins . A systematic factorial design would allow researchers to identify optimal conditions for soluble Y57E12AM.1 expression, potentially achieving yields comparable to the 250 mg/L reported for other recombinant proteins using similar approaches .

How can the structural integrity and functional activity of recombinant Y57E12AM.1 be verified?

Verification of structural integrity and functional activity should employ multiple complementary techniques:

• SDS-PAGE and Western blotting: To confirm protein size, purity (>90%), and identity using specific antibodies against Y57E12AM.1 or the His-tag

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): To determine the oligomeric state and accurate molecular weight in solution, accounting for post-translational modifications

• Circular Dichroism (CD) Spectroscopy: To assess secondary structure content, particularly important for transmembrane proteins to confirm proper folding

• Functional Assays: While specific assays for Y57E12AM.1 are not well-documented in the provided sources, binding assays using surface plasmon resonance (similar to those used for other recombinant proteins) could be adapted if interaction partners are identified

What approaches can be used to study Y57E12AM.1 function in C. elegans?

To investigate Y57E12AM.1 function in C. elegans, researchers can employ:

• RNA interference (RNAi): Knockdown of Y57E12AM.1 expression through feeding, injection, or soaking methods followed by phenotypic analysis

• CRISPR-Cas9 gene editing: Generation of knockout or tagged knock-in strains to study loss-of-function phenotypes or protein localization

• Tissue-specific expression: Rescue experiments in mutant backgrounds using tissue-specific promoters to identify where Y57E12AM.1 function is required

• Interaction studies: Yeast two-hybrid or co-immunoprecipitation experiments to identify protein interaction partners, which may provide insights into Y57E12AM.1 function

• Subcellular localization: Immunofluorescence or expression of fluorescently-tagged Y57E12AM.1 to determine cellular distribution

Given that Y57E12AM.1 has been annotated as potentially involved in oligosaccharyl transferase activity (based on its synonyms) , assays that measure glycosylation efficiency could be particularly informative.

What structural analysis techniques are most suitable for Y57E12AM.1 characterization?

As a transmembrane protein, Y57E12AM.1 presents specific challenges for structural characterization. The following approaches can be considered:

• X-ray crystallography: Requires optimization of crystallization conditions for membrane proteins, potentially using lipidic cubic phase methods

• Cryo-electron microscopy (cryo-EM): Particularly useful for membrane proteins that resist crystallization

• Nuclear Magnetic Resonance (NMR) spectroscopy: Suitable for determining structure of smaller membrane proteins or domains in membrane-mimetic environments

• Computational modeling: Homology modeling based on related proteins with known structures, followed by molecular dynamics simulations to predict structural dynamics

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To probe protein dynamics and solvent accessibility

The relatively small size of Y57E12AM.1 (79 amino acids) makes it potentially amenable to solution NMR studies if suitable membrane mimetics can be identified.

How might Y57E12AM.1 function in the context of protein glycosylation pathways?

Based on its annotation as "Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit" and "Oligosaccharyl transferase subunit" , Y57E12AM.1 likely functions in the N-linked glycosylation pathway. To investigate this:

• Glycoprotein analysis: Compare glycosylation patterns in wild-type versus Y57E12AM.1-deficient C. elegans using mass spectrometry

• In vitro glycosylation assays: Reconstitute purified Y57E12AM.1 with other oligosaccharyl transferase components to assess enzymatic activity

• Co-expression studies: Express Y57E12AM.1 with other components of the oligosaccharyl transferase complex to study complex formation and activity

• Evolutionary conservation analysis: Compare Y57E12AM.1 sequence and function with homologs in other species to identify conserved functional domains

This research direction would contribute significantly to understanding the role of Y57E12AM.1 in protein glycosylation, a critical post-translational modification in eukaryotes.

What strategies can address poor solubility of recombinant Y57E12AM.1?

As a transmembrane protein, Y57E12AM.1 may present solubility challenges. Consider these approaches:

• Fusion tags: Beyond His-tags, solubility-enhancing tags like SUMO, MBP, or GST may improve soluble expression

• Detergent screening: Systematic screening of detergents (non-ionic, zwitterionic, etc.) to identify optimal solubilization conditions

• Expression as membrane protein fragments: Express soluble domains separately if full-length protein proves recalcitrant

• Co-expression with chaperones: GroEL/GroES or other chaperone systems can improve folding of challenging proteins

• Nanodiscs or amphipols: These membrane-mimetic systems can stabilize transmembrane proteins in a native-like environment

How can researchers assess the functional significance of Y57E12AM.1 in C. elegans development?

To evaluate the developmental role of Y57E12AM.1:

• Developmental timing analysis: Monitor developmental milestones in Y57E12AM.1 mutants or RNAi-treated animals

• Tissue-specific rescue experiments: Determine in which tissues Y57E12AM.1 function is required during development

• Glycoprotein profiling during development: Assess glycosylation changes in developing C. elegans with altered Y57E12AM.1 function

• Genetic interaction studies: Conduct screens for enhancers or suppressors of Y57E12AM.1 mutant phenotypes

• Transcriptomic analysis: Compare gene expression patterns between wild-type and Y57E12AM.1-deficient animals during development

These approaches would provide insights into both the biochemical function and developmental significance of Y57E12AM.1 in C. elegans.