Recombinant UPF0266 membrane protein YPTB1631 (YPTB1631)

How is recombinant YPTB1631 typically produced for research applications?

The recombinant YPTB1631 protein is typically produced in E. coli expression systems with an N-terminal His-tag to facilitate purification. The expression construct contains the full-length coding sequence (amino acids 1-153) of the native protein . When designing expression strategies, researchers should consider that:

• The protein is membrane-associated, which can complicate expression and purification

• E. coli BL21(DE3) or similar strains are commonly used as expression hosts

• The His-tag enables purification via immobilized metal affinity chromatography (IMAC)

• The purified protein typically achieves >90% purity as determined by SDS-PAGE

What are the optimal storage conditions for recombinant YPTB1631?

Optimal storage of recombinant YPTB1631 requires careful attention to temperature and formulation:

• Store lyophilized protein at -20°C/-80°C upon receipt

• For reconstituted protein, aliquot to avoid repeated freeze-thaw cycles

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

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is recommended)

• Store glycerol-containing aliquots at -20°C/-80°C

Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided to maintain structural integrity and function .

What is the recommended protocol for reconstituting lyophilized YPTB1631?

For optimal reconstitution of lyophilized YPTB1631:

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

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

• For long-term storage, add glycerol to a final concentration of 5-50%

• Aliquot to minimize freeze-thaw cycles

• The protein is typically supplied in a Tris/PBS-based buffer with 6% trehalose at pH 8.0

This reconstitution approach helps maintain protein stability and function while minimizing aggregation or precipitation that can occur with membrane proteins.

What experimental approaches can be used to optimize YPTB1631 expression in E. coli systems?

Optimizing membrane protein expression requires systematic evaluation of multiple variables. Based on comparable studies with other recombinant proteins, a factorial experimental design approach can be highly effective:

• Evaluate induction parameters:

  • IPTG concentration (typically 0.1-1.0 mM)

  • Induction temperature (15-37°C, with lower temperatures often favoring proper folding)

  • Induction duration (4-24 hours)

  • Induction at different culture densities (OD600 of 0.6-1.0)

• Optimize media composition:

  • Compare rich media (LB, TB, 2YT) vs. minimal media

  • Test supplementation with glucose (0.5-1%)

  • Evaluate additives that can enhance membrane protein expression:

    • Glycerol (0.5-2%)

    • Sorbitol (0.5-1 M)

    • Betaine (1-2.5 mM)

• Consider expression strains specifically designed for membrane proteins:

  • C41(DE3) and C43(DE3)

  • Lemo21(DE3)

  • Rosetta strains for rare codon optimization

Statistical analysis of these variables can identify optimal conditions, as demonstrated in comparable studies where expression increased from minimal to 250 mg/L through systematic optimization .

What strategies can address common challenges in purifying membrane proteins like YPTB1631?

Membrane protein purification presents several challenges that can be addressed with specialized techniques:

• Solubilization strategies:

  • Screen multiple detergents (DDM, LDAO, OG, CHAPS) at various concentrations

  • Test mixed micelle systems combining different detergent types

  • Consider nanodiscs or amphipols for maintaining native-like environment

• Two-step purification approach:

  • Initial IMAC purification using the His-tag

  • Secondary purification via size exclusion chromatography or ion exchange

  • Target >90% purity as verified by SDS-PAGE

• Protein stabilization:

  • Maintain detergent concentration above CMC throughout purification

  • Include glycerol (10-20%) in all buffers

  • Test stabilizing additives (specific lipids, cholesterol hemisuccinate)

  • Optimize pH and ionic strength

• Quality control:

  • Verify protein folding through circular dichroism or fluorescence spectroscopy

  • Assess homogeneity by dynamic light scattering

  • Confirm functionality through binding or activity assays specific to the protein family

How can researchers investigate the potential functions of YPTB1631 given its uncharacterized status?

Investigating uncharacterized membrane proteins like YPTB1631 requires multiple complementary approaches:

• Bioinformatic analysis:

  • Sequence homology with characterized proteins across species

  • Structural prediction using tools like AlphaFold2

  • Identification of conserved domains or motifs

  • Genomic context analysis (operons, neighboring genes)

• Protein interaction studies:

  • Pull-down assays using the His-tagged protein

  • Bacterial two-hybrid screening

  • Crosslinking mass spectrometry to identify interaction partners

  • Co-immunoprecipitation with candidate interactors

• Gene knockout/complementation:

  • Phenotypic analysis of YPTB1631 deletion mutants

  • Complementation studies with the recombinant protein

  • Comparative transcriptomics of wild-type vs. mutant strains

• Localization studies:

  • Immunofluorescence microscopy

  • Subcellular fractionation followed by Western blotting

  • GFP fusion protein analysis

These approaches can provide converging evidence about the functional role of this uncharacterized membrane protein in Yersinia pseudotuberculosis biology.

What techniques can be used to analyze the membrane topology of YPTB1631?

Understanding membrane protein topology is crucial for functional studies. For YPTB1631, consider these methods:

• Computational prediction:

  • Hydropathy analysis of the amino acid sequence

  • Topology prediction algorithms (TMHMM, Phobius, TOPCONS)

  • Comparison with homologous proteins of known topology

• Experimental verification:

  • Cysteine scanning mutagenesis with sulfhydryl reagents

  • Protease protection assays

  • Fluorescence resonance energy transfer (FRET)

  • Epitope insertion followed by accessibility studies

• Advanced structural approaches:

  • Cryo-electron microscopy

  • X-ray crystallography (challenging but potentially informative)

  • Solid-state NMR spectroscopy

Combining computational predictions with experimental validation provides the most reliable topology model.

How might genetic variations in YPTB1631 impact protein function and research applications?

Analyzing genetic variations requires consideration of both natural variants and experimental mutations:

• Natural variation analysis:

  • Compare YPTB1631 sequences across Yersinia strains

  • Identify conserved vs. variable regions

  • Correlate variations with phenotypic differences between strains

• Site-directed mutagenesis approaches:

  • Target conserved amino acids to identify essential residues

  • Modify predicted functional domains

  • Create chimeric proteins with homologs to map functional regions

• Structural impact assessment:

  • Model the effect of variations on protein folding

  • Identify mutations that might affect membrane insertion

  • Predict alterations to protein-protein interaction interfaces

This understanding helps contextualize experimental results and can guide the design of protein variants with altered functions for mechanism studies.

What is known about the transcriptional regulation of YPTB1631 and how might this inform research applications?

Understanding the transcriptional regulation provides context for protein expression studies:

• Promoter analysis:

  • Identify transcription factor binding sites in the promoter region

  • Analyze the presence of regulatory elements like UTR sequences that might affect translation

  • Consider similarities to known regulated genes in Yersinia

• Expression analysis methods:

  • qRT-PCR under different growth conditions

  • RNA-seq data mining across various experimental conditions

  • Reporter gene fusions to monitor promoter activity

• Regulatory network integration:

  • Identify conditions that upregulate or downregulate expression

  • Connect expression patterns to specific stress responses or virulence pathways

  • Consider potential post-transcriptional regulation mechanisms

Research with recombinant proteins should ideally reflect physiologically relevant conditions for expression that might be informed by understanding these regulatory mechanisms.

What are the challenges and strategies for crystallization of membrane proteins like YPTB1631?

Membrane protein crystallization presents unique challenges requiring specialized approaches:

• Detergent screening:

  • Systematic testing of detergent types and concentrations

  • Evaluating detergent-lipid mixtures

  • Using facial amphiphiles or novel surfactants

• Crystallization techniques:

  • Lipidic cubic phase (LCP) crystallization

  • Bicelle-based crystallization

  • Vapor diffusion with specialized additives

  • Antibody fragment co-crystallization to provide crystal contacts

• Protein engineering for crystallization:

  • Truncation of flexible regions

  • Fusion with crystallization chaperones

  • Surface entropy reduction through targeted mutations

  • Thermostabilizing mutations

• Alternative structural approaches:

  • Cryo-EM single-particle analysis

  • Electron crystallography

  • NMR studies of selectively labeled protein

These methods require iterative optimization but can provide critical structural insights unavailable through other approaches.

How does YPTB1631 compare to homologous proteins in other bacterial species, and what can this tell us about its function?

Comparative analysis provides evolutionary context and functional hints:

• Homology search strategies:

  • BLAST analysis against bacterial proteomes

  • Hidden Markov Model profile searches

  • Structure-based similarity searches

• Phylogenetic analysis:

  • Construction of phylogenetic trees to identify closest homologs

  • Mapping of conserved vs. divergent regions

  • Correlation with bacterial taxonomy and ecological niches

• Functional inference:

  • Data mining of characterized homologs in model organisms

  • Identification of conserved functional motifs

  • Analysis of genomic context conservation (synteny)

• Experimental validation:

  • Complementation studies across species

  • Heterologous expression and functional testing

  • Comparison of interaction partners between homologs

This comparative approach can provide crucial insights when direct experimental data on YPTB1631 is limited.

What are the most promising research directions for further characterizing YPTB1631?

Future research on YPTB1631 could focus on:

• Functional genomics approaches:

  • CRISPR-Cas9 based gene editing in Yersinia

  • High-throughput phenotypic screening

  • Suppressor mutation analysis

• Systems biology integration:

  • Proteomics to identify interaction networks

  • Metabolomics to detect metabolic changes in mutants

  • Multi-omics data integration

• Structural biology advancements:

  • Cryo-EM single-particle analysis

  • Hydrogen-deuterium exchange mass spectrometry

  • Solid-state NMR spectroscopy

• Translational research potential:

  • Evaluation as a potential therapeutic target

  • Development of specific antibodies or inhibitors

  • Assessment of role in bacterial virulence

These approaches can collectively advance our understanding of this uncharacterized membrane protein and potentially reveal new insights into Yersinia pseudotuberculosis biology and pathogenesis.