Recombinant UPF0272 protein WS0139 (WS0139)

What is UPF0272 protein WS0139 and what organism does it originate from?

UPF0272 protein WS0139 is a protein of currently unknown function (as indicated by the UPF designation) derived from the bacterium Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W), formerly known as Vibrio succinogenes. The protein is identified in the UniProt database with accession number Q7MSU1 . Understanding the evolutionary origin of this protein provides important context for functional studies, particularly when conducting comparative analyses with homologous proteins from related bacterial species.

What expression systems are recommended for producing recombinant WS0139?

For recombinant production of WS0139, a baculovirus expression system has been successfully employed as indicated in the product documentation . When designing expression experiments, researchers should consider that:

• The baculovirus system is advantageous for expressing prokaryotic proteins that require post-translational modifications

• The full-length protein (expression region 1-396) can be successfully expressed using this system

• Alternative expression systems (E. coli, yeast, mammalian) may be attempted depending on experimental requirements, though optimization would be necessary

When adapting to different expression systems, codon optimization should be considered to enhance expression efficiency in the host organism.

What are the optimal storage conditions for maintaining WS0139 stability?

Proper storage of recombinant WS0139 is crucial for maintaining its structural integrity and biological activity. The recommended storage conditions depend on the formulation:

For working solutions, it is advisable to store aliquots at 4°C for no more than one week. Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of activity . Researchers should monitor protein stability over time using appropriate assays (e.g., SDS-PAGE, activity assays) to verify the retention of structural integrity and function.

What is the recommended reconstitution protocol for lyophilized WS0139?

For optimal reconstitution of lyophilized WS0139:

• Centrifuge the vial briefly before opening to ensure the protein pellet is at 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 the default recommendation)

• Prepare small aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C to -80°C for long-term storage

The addition of glycerol serves as a cryoprotectant to prevent protein denaturation during freezing. The specific glycerol concentration may require optimization depending on downstream applications, as high glycerol concentrations may interfere with some assays.

How can researchers assess the purity and integrity of WS0139 preparations?

The standard purity of commercial WS0139 preparations is >85% as determined by SDS-PAGE . Researchers should independently verify purity and integrity through:

• SDS-PAGE analysis: Run both reducing and non-reducing conditions to assess potential disulfide bonding

• Western blot analysis: If antibodies are available, use them to confirm identity

• Mass spectrometry: For precise molecular weight determination and peptide mapping

• Size exclusion chromatography: To assess aggregation state and homogeneity

• Dynamic light scattering: To evaluate size distribution and potential aggregation

A comprehensive purity assessment is essential before proceeding with functional studies to ensure that observed activities are attributable to WS0139 rather than contaminants.

What structural analysis techniques are most suitable for characterizing WS0139?

Given the unknown function of UPF0272 protein WS0139, structural characterization is crucial. Recommended techniques include:

Combining multiple techniques provides complementary structural information that can guide functional hypotheses. Since the protein contains multiple cysteine residues, disulfide mapping may also be informative for understanding structural stabilization.

What approaches can be used to investigate potential binding partners of WS0139?

For a protein of unknown function like WS0139, identifying interaction partners is a critical step toward functional characterization. Recommended methods include:

• Pull-down assays: Using tagged WS0139 as bait to capture binding partners from cell lysates

• Yeast two-hybrid screening: For detecting binary protein-protein interactions

• Biolayer interferometry or surface plasmon resonance: For measuring binding kinetics with candidate partners

• Co-immunoprecipitation: When antibodies are available

• Crosslinking mass spectrometry: To identify interacting protein regions

• Proximity labeling (BioID, APEX): For identifying proteins in close proximity in cellular contexts

Candidate binding partners should be selected based on genomic context analysis and co-expression patterns in the native organism, Wolinella succinogenes.

How can researchers probe the potential enzymatic activity of WS0139?

Since UPF0272 family proteins have unknown functions, a systematic approach to enzymatic activity screening is recommended:

• Sequence-based prediction: Use tools like InterProScan, PFAM, and PROSITE to identify potential functional domains

• Structure-based prediction: If structural data becomes available, tools like ProFunc and COACH can predict active sites

• Activity screening panels: Test for common enzymatic activities (hydrolase, transferase, kinase, etc.)

• Metabolite profiling: Compare metabolite profiles in systems with and without WS0139 expression

• Genetic context analysis: Examine the genomic neighborhood in W. succinogenes for functional clues

Researchers should also consider the physiological context of W. succinogenes as an anaerobic, fumarate-respiring bacterium when designing activity assays.

What expression tag systems are compatible with WS0139 for purification and detection?

When designing constructs for WS0139 expression:

• Tag selection considerations:

  • The tag type for commercial preparations is determined during the manufacturing process

  • N-terminal tags may be preferable if C-terminal folding is critical

  • C-terminal tags may be preferable if the N-terminus contains signal sequences

• Common compatible tags:

  • Affinity tags: His6, GST, MBP, FLAG

  • Solubility-enhancing tags: SUMO, TRX, NusA

  • Detection tags: GFP, RFP, Luciferase

• Tag removal strategies:

  • TEV, PreScission, or Thrombin protease sites can be incorporated

  • Factor Xa recognition sites may be considered for specific applications

The impact of tags on protein solubility, activity, and structure should be carefully evaluated through comparative studies with tagged and untagged versions when possible.

What control experiments should be included when studying WS0139 function?

Rigorous experimental design for studying a protein of unknown function like WS0139 should include:

• Negative controls:

  • Buffer-only conditions

  • Irrelevant protein of similar size/structure

  • Heat-denatured WS0139

• Positive controls:

  • Known proteins with similar predicted domains

  • Commercially available enzyme standards for activity assays

• Validation approaches:

  • Multiple experimental techniques confirming the same result

  • Concentration-dependent responses

  • Mutations of predicted key residues

  • Complementation studies in knockout systems

Careful control design is essential for distinguishing true biological functions from artifacts, particularly for proteins of unknown function.

How can researchers design experiments to elucidate the physiological role of WS0139?

To investigate the physiological significance of WS0139:

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 or homologous recombination in W. succinogenes

  • Analysis of phenotypic changes under various growth conditions

• Complementation assays:

  • Reintroduction of wild-type and mutant WS0139 into knockout strains

  • Cross-species complementation with homologs

• Expression profiling:

  • RT-qPCR or RNA-seq to determine expression patterns under different conditions

  • Promoter reporter fusions to monitor expression

• Localization studies:

  • Fluorescent protein fusions or immunolocalization

  • Subcellular fractionation followed by Western blotting

• Interactome mapping:

  • Systematic identification of protein interaction networks

  • Genetic interaction screens

These approaches should be integrated with biochemical characterization to form a comprehensive understanding of WS0139's role in W. succinogenes physiology.

What are common challenges when working with WS0139 and how can they be addressed?

Researchers working with recombinant proteins like WS0139 often encounter several technical challenges:

Detailed laboratory records documenting protein behavior under various conditions can be invaluable for troubleshooting and protocol optimization.

How can researchers validate antibodies or develop detection methods for WS0139?

For specific detection of WS0139 in complex samples:

• Antibody development and validation:

  • Generate polyclonal antibodies against purified WS0139

  • Validate specificity using Western blotting against recombinant WS0139

  • Confirm specificity using knockout/knockdown controls

  • Test cross-reactivity with homologous proteins

• Alternative detection methods:

  • Mass spectrometry-based targeted proteomics (SRM/MRM)

  • Aptamer development for specific binding

  • Activity-based protein profiling if enzymatic function is identified

• Epitope mapping:

  • Identify immunogenic regions for more specific antibody production

  • Design peptide-specific antibodies targeting unique regions

Proper validation using positive and negative controls is essential for ensuring detection specificity and sensitivity.