Recombinant Yersinia pestis UPF0299 membrane protein YPDSF_1460 (YPDSF_1460)

What is Yersinia pestis UPF0299 membrane protein YPDSF_1460?

Yersinia pestis UPF0299 membrane protein YPDSF_1460 is a full-length protein (135 amino acids) found in the membrane of Yersinia pestis, the causative agent of plague. The protein belongs to the UPF0299 family of uncharacterized proteins, with the "UPF" designation indicating that its function remains incompletely understood. The protein has been assigned the UniProt ID A4TKN5 and is predicted to be involved in membrane-related functions based on its localization and structural characteristics .

The protein's properties indicate that it is an integral membrane protein with multiple transmembrane domains, as suggested by its hydrophobic amino acid composition and predicted secondary structure. When produced recombinantly, YPDSF_1460 is typically expressed with a His-tag to facilitate purification and subsequent experimental analysis .

What are the recommended storage conditions for purified YPDSF_1460?

For optimal stability and activity maintenance of purified recombinant YPDSF_1460 protein, the following storage conditions are recommended:

• Store the lyophilized powder at -20°C to -80°C upon receipt

• After reconstitution, 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% (with 50% being optimal) and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles as they can lead to protein degradation and loss of activity

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

• Prior to opening, briefly centrifuge the vial to bring contents to the bottom

These storage recommendations are based on empirical observations for maintaining protein stability and function. Proper aliquoting is essential to minimize freeze-thaw cycles, and the addition of glycerol serves as a cryoprotectant to prevent ice crystal formation that can damage protein structure.

What expression systems are most effective for YPDSF_1460 production?

Escherichia coli is the most commonly used and effective expression system for recombinant YPDSF_1460 production. Several factors contribute to this choice:

• E. coli's rapid growth and high protein yields make it suitable for membrane protein expression when properly optimized

• The availability of specialized E. coli strains designed for membrane protein expression (e.g., C41(DE3), C43(DE3), or Lemo21(DE3))

• Compatibility with various fusion tags, particularly N-terminal His-tags that facilitate purification

To optimize expression in E. coli:

• Use lower growth temperatures (16-25°C) to reduce inclusion body formation

• Consider induction with lower concentrations of IPTG (0.1-0.5 mM)

• Implement longer expression times (16-24 hours) at reduced temperatures

• Add glycylbetaine or other osmolytes to the growth medium to stabilize membrane proteins

• Evaluate different E. coli strains engineered for membrane protein expression

Alternative expression systems such as yeast (Pichia pastoris) or insect cells may be considered if functional studies require eukaryotic post-translational modifications, though these systems typically yield lower amounts of protein compared to E. coli .

How can I improve translation initiation efficiency for YPDSF_1460 expression?

Improving translation initiation is critical for successful recombinant protein expression, especially for membrane proteins like YPDSF_1460. Research has shown that the accessibility of translation initiation sites is a key determinant of expression success. To optimize this aspect:

• Evaluate and modify the mRNA secondary structure around the translation initiation site using computational tools that model base-unpairing across the Boltzmann's ensemble

• Implement synonymous codon substitutions in the first nine codons of the mRNA to improve ribosomal binding site accessibility

• Consider using the TIsigner tool, which uses simulated annealing to optimize the translation initiation region through synonymous substitutions

• Remove rare codons near the translation start site that might impede the initiation process

• Balance expression optimization with cell growth, as higher accessibility can lead to higher protein production but slower cell growth

Studies analyzing 11,430 recombinant proteins have demonstrated that accessibility of translation initiation sites significantly outperforms alternative features in predicting expression success. This approach can be particularly valuable for challenging membrane proteins like YPDSF_1460, where even modest modifications to the coding sequence can substantially improve expression levels .

What purification strategies yield the highest purity for YPDSF_1460?

For obtaining high-purity recombinant YPDSF_1460 protein, a multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using the N-terminal His-tag

  • Use Ni-NTA or Co-NTA resins with imidazole gradients (20-250 mM) for elution

  • Include detergents (0.1% DDM or 0.5% CHAPS) in buffers to maintain protein solubility

• Intermediate purification: Size exclusion chromatography (SEC)

  • Separates protein based on size, removing aggregates and contaminating proteins

  • Provides information about oligomeric state of the protein

• Polishing step: Ion exchange chromatography

  • Further removes contaminants based on charge differences

  • Select appropriate resin (anion/cation) based on protein's theoretical pI

• Quality control measurements:

  • SDS-PAGE analysis (aim for >90% purity)

  • Western blot confirmation using anti-His antibodies

  • Mass spectrometry verification

For YPDSF_1460 specifically, maintaining the proper detergent concentration throughout the purification process is critical to prevent protein aggregation and maintain the native membrane protein conformation. Additionally, inclusion of glycerol (5-10%) in all purification buffers helps stabilize the protein .

How should I design experiments to investigate YPDSF_1460 function?

When designing experiments to investigate the function of YPDSF_1460, a systematic approach combining multiple experimental paradigms is recommended:

• Start with a full factorial design approach:

  • Identify key experimental factors (e.g., pH, temperature, ligand concentration)

  • Include all possible combinations of factor levels to identify interactions

  • For example, a 3×2 design might explore three protein concentrations at two different pH levels

• Consider within-subject vs. between-subject designs:

  • For biochemical assays, repeated measurements on the same protein preparation (within-subject)

  • For cell-based assays, comparing different cell lines or treatments (between-subject)

• Sample size determination:

  • Calculate adequate replication based on expected effect size and desired statistical power

  • Generally, aim for at least 3-5 biological replicates and 2-3 technical replicates per condition

• Specific experimental approaches for membrane proteins:

  • Membrane localization studies using fluorescent protein fusions

  • Protein-protein interaction studies using bacterial two-hybrid systems

  • Liposome reconstitution experiments to assess transport or channel functions

  • Site-directed mutagenesis to identify functional residues

• Controls to include:

  • Empty vector control for expression studies

  • Inactive mutant versions (e.g., point mutations in predicted functional domains)

  • Related membrane proteins from non-pathogenic Yersinia species

When analyzing results, implement appropriate statistical approaches based on the experimental design, such as two-way ANOVA for factorial designs with multiple factors .

What techniques are most suitable for studying YPDSF_1460 membrane localization and topology?

To investigate the membrane localization and topology of YPDSF_1460, several complementary techniques should be employed:

A comprehensive experimental workflow would begin with computational predictions, followed by reporter fusion experiments to validate the predicted topology. Subsequent biochemical approaches can further refine the model, with structural biology techniques providing more detailed information. When designing fusion constructs, care must be taken to avoid disrupting transmembrane domains, which could lead to mislocalization or misfolding of the protein .

How can I assess the role of YPDSF_1460 in Yersinia pestis virulence?

Investigating the role of YPDSF_1460 in Yersinia pestis virulence requires a multi-faceted approach combining genetic, cellular, and in vivo methodologies:

• Genetic approaches:

  • Generate clean deletion mutants (ΔYPDSF_1460) using allelic exchange

  • Create complemented strains with wild-type and mutant versions

  • Develop conditional knockdown strains for essential genes using inducible systems

• In vitro virulence assays:

  • Measure bacterial survival in macrophages and neutrophils

  • Assess resistance to specific host defense mechanisms (oxidative stress, antimicrobial peptides)

  • Evaluate biofilm formation capacity

• Transcriptomic and proteomic analyses:

  • RNA-seq comparing wild-type and mutant strains under virulence-inducing conditions

  • Proteomics to identify interaction partners and affected pathways

  • Phosphoproteomics to detect changes in signaling cascades

• Animal model studies (requiring appropriate biosafety facilities):

  • Virulence assessment in mouse models of bubonic and pneumonic plague

  • Competitive index experiments comparing wild-type and mutant strains

  • Tissue bacterial burden and histopathological analyses

• Experimental design considerations:

  • Implement full factorial designs to test multiple variables (e.g., temperature, growth phase)

  • Include appropriate controls (wild-type, known virulence factor mutants)

  • Use sufficient biological replicates (n≥5 for animal studies)

How does YPDSF_1460 compare structurally and functionally to homologs in other Yersinia species?

Comparative analysis of YPDSF_1460 with homologs in other Yersinia species provides valuable insights into evolutionary conservation and potential functional importance:

The high sequence conservation between Y. pestis and Y. pseudotuberculosis homologs reflects their close evolutionary relationship, while more significant differences exist with Y. enterocolitica homologs. The analysis of these differences, particularly in transmembrane domains, can provide insights into species-specific adaptations and potential roles in virulence.

Research methodologies for comparative analysis should include:

• Sequence alignment and phylogenetic analysis to establish evolutionary relationships

• Homology modeling to predict structural differences

• Heterologous expression of homologs to compare biochemical properties

• Complementation studies in knockout strains to assess functional equivalence

• Domain swapping experiments to identify regions responsible for functional differences

This comparative approach is particularly valuable for understanding how YPDSF_1460 may contribute to the unique virulence properties of Y. pestis compared to other Yersinia species that cause different disease manifestations.

What methodologies are most effective for determining the structure-function relationship of YPDSF_1460?

Elucidating the structure-function relationship of YPDSF_1460 requires an integrated approach combining computational predictions, molecular biology techniques, and structural analyses:

• Computational approaches:

  • Secondary structure prediction using PSIPRED, JPred

  • Homology modeling using Phyre2, I-TASSER, or AlphaFold2

  • Molecular dynamics simulations to predict conformational changes

  • Identification of conserved motifs through multiple sequence alignments

• Directed mutagenesis strategies:

  • Alanine scanning of predicted functional residues

  • Cysteine scanning for accessibility and disulfide mapping

  • Conservative and non-conservative substitutions of key residues

  • Construction of chimeric proteins with homologs from other species

• Functional assays:

  • Liposome reconstitution for transport studies

  • Circular dichroism to assess secondary structure changes

  • Thermal shift assays to evaluate protein stability

  • Surface plasmon resonance for interaction studies

• Structural biology techniques:

  • X-ray crystallography of detergent-solubilized protein

  • Cryo-electron microscopy for membrane protein complexes

  • NMR for dynamic regions and smaller domains

  • Cross-linking mass spectrometry for domain-domain interactions

• Experimental design considerations:

  • Implement systematic mutation strategy rather than random approaches

  • Use factorial design to test multiple variables simultaneously

  • Include appropriate controls for each experimental technique

  • Correlate structural predictions with functional outcomes

One particularly effective approach is to combine site-directed mutagenesis with functional assays. By systematically mutating residues predicted to be important based on computational analyses, and then assessing the impact on protein function, researchers can build a comprehensive map of structure-function relationships for YPDSF_1460.

How can advanced expression optimization techniques improve YPDSF_1460 yield and quality?

Optimizing the expression of membrane proteins like YPDSF_1460 presents unique challenges that can be addressed through several advanced techniques:

• Codon optimization strategies:

  • Focus on translation initiation site accessibility rather than simple codon adaptation

  • Implement the TIsigner approach to modify the first nine codons with synonymous substitutions

  • Balance GC content and avoid rare codon clusters

  • Consider mRNA secondary structure predictions to minimize stable structures near the start codon

• Expression vector engineering:

  • Test multiple fusion tags (His, MBP, SUMO, TrxA) for improved solubility

  • Evaluate different promoter strengths (T7, tac, araBAD)

  • Include translation enhancing elements like SUMO or MBP

  • Incorporate inducible promoters with tight regulation

• Host strain optimization:

  • Screen specialized E. coli strains designed for membrane proteins (C41/C43(DE3), Lemo21)

  • Consider strains with altered membrane composition (PE-deficient strains)

  • Test strains with additional chaperones or foldases

  • Evaluate strains with reduced proteolytic activity

• Process optimization parameters:

  Parameter	Standard Condition	Optimized Range	Effect on Yield
  Temperature	37°C	16-25°C	2-3× improvement
  Inducer concentration	1 mM IPTG	0.1-0.5 mM IPTG	1.5-2× improvement
  Media composition	LB	TB, 2×YT, autoinduction	2-4× improvement
  Additives	None	Glycerol, sucrose, betaine	1.5-3× improvement
  Growth phase at induction	Mid-log	Late-log	1.2-1.8× improvement

• High-throughput screening approaches:

  • Design of experiments (DoE) methodology to efficiently test multiple parameters

  • Parallel small-scale expression trials with factorial design

  • Fluorescent reporter fusions for rapid detection of well-folded protein

  • Automated purification screening to assess quality and yield simultaneously

Remember that the accessibility of translation initiation sites has been shown to significantly outperform alternative features in predicting expression success. Research analyzing 11,430 recombinant proteins demonstrated that modest synonymous changes are sufficient to tune recombinant protein expression levels, supporting the idea that a higher accessibility leads to higher protein production, albeit potentially with slower cell growth .

What are the emerging techniques for studying the interaction of YPDSF_1460 with the bacterial membrane?

Several cutting-edge techniques are emerging for studying membrane protein interactions that can be applied to YPDSF_1460:

• Advanced microscopy approaches:

  • Super-resolution microscopy (STORM, PALM) for in situ localization

  • High-speed atomic force microscopy for dynamic interactions

  • Correlative light and electron microscopy for combining functional and structural data

  • Single-molecule tracking to monitor diffusion and clustering

• Lipid-protein interaction techniques:

  • Native nanodiscs for maintaining native lipid environment

  • Hydrogen-deuterium exchange mass spectrometry for identifying lipid-interacting regions

  • Lipid pull-down assays with synthetic liposomes of defined composition

  • Fluorescence anisotropy for measuring protein-lipid binding kinetics

• Membrane mimetic systems:

  • Cell-free expression directly into nanodiscs or liposomes

  • Polymer-based membrane mimetics (SMALPs, amphipols)

  • Droplet interface bilayers for functional studies

  • Microfluidic systems for high-throughput screening

• Computational approaches:

  • Coarse-grained molecular dynamics simulations for long timescale events

  • Machine learning prediction of lipid-binding sites

  • Multiscale modeling combining atomistic and mesoscale simulations

  • Molecular docking with flexible membrane components

• Experimental design considerations:

  • Implement factorial designs to test protein interactions with different lipid compositions

  • Consider time-resolved experiments to capture dynamic interactions

  • Combine multiple techniques to validate findings across different experimental systems

  • Use appropriate controls including non-membrane proteins and scrambled lipid compositions

These emerging techniques offer unprecedented resolution and insight into how membrane proteins like YPDSF_1460 interact with their lipid environment. By combining these approaches, researchers can develop a comprehensive understanding of how the protein functions within the bacterial membrane and potentially identify novel targets for therapeutic intervention.