Recombinant Pseudomonas aeruginosa UPF0060 membrane protein PSPA7_1846 (PSPA7_1846)

What is Pseudomonas aeruginosa UPF0060 membrane protein PSPA7_1846?

Pseudomonas aeruginosa UPF0060 membrane protein PSPA7_1846 is a full-length membrane protein found in Pseudomonas aeruginosa strain PA7. It belongs to the UPF0060 protein family and consists of 109 amino acids with the sequence: MINYLWFVLAAFCEIAGCYAFYLWLRLGKSALWVLPGLLSLSLFALLLTRVEASYAGRAYAAYGGIYVAASLFWLAFVERSRPLWSDWLGVALCVLGASIVLFGPRLSQ . The protein has a UniProt accession number of A6V2E4 and is characterized by its transmembrane domains that integrate into the cell membrane . As a full-length protein, it contains the complete amino acid sequence from the N-terminal to the C-terminal, which is essential for understanding its biological functions within the bacterial cell membrane structure and potential pathogenic roles.

How does the structure of PSPA7_1846 relate to its membrane localization?

The PSPA7_1846 protein contains hydrophobic regions within its amino acid sequence that facilitate its integration into the bacterial cell membrane. Analysis of its primary structure reveals multiple hydrophobic stretches that likely form transmembrane domains spanning the phospholipid bilayer. These regions include the segments "MINYLWFVLAAFCEIAGCYAFY" and "LWLRLGKSALWVLPGLLSLSLFALLLT" which demonstrate characteristic hydrophobicity patterns typical of membrane-spanning domains . The protein's membrane localization is further supported by the presence of charged residues at boundary regions that likely interact with the polar head groups of membrane phospholipids. Understanding this structural arrangement is crucial for designing experiments involving protein extraction, purification, and functional characterization, as membrane proteins require specialized handling to maintain their native conformation and activity.

What are the optimal expression systems for recombinant PSPA7_1846 production?

The optimal expression strategy should include:

For PSPA7_1846, which is a bacterial membrane protein, an E. coli system with specialized strains designed for membrane protein expression often provides the best balance between yield and functionality .

What fusion tags are recommended for improving PSPA7_1846 solubility and purification?

Selection of appropriate fusion tags is critical for successful expression and purification of membrane proteins like PSPA7_1846. Several fusion partners can enhance solubility, expression, and purification efficiency:

For PSPA7_1846, a dual tagging approach with an N-terminal solubility enhancer (MBP or SUMO) combined with a C-terminal His-tag often provides optimal results for expression validation, purification monitoring, and maintaining protein stability . The chosen tag should be selected based on the specific experimental requirements, with consideration for whether the tag needs to be removed for downstream applications such as structural studies or functional assays .

How should researchers design experiments to study PSPA7_1846 function?

When designing experiments to investigate PSPA7_1846 function, researchers should follow systematic experimental design principles. First, clearly define the variables involved: the independent variable (e.g., PSPA7_1846 expression levels, mutation status, or environmental conditions) and the dependent variable (e.g., bacterial virulence, membrane integrity, or protein-protein interactions) . Formulate a specific, testable hypothesis based on computational predictions or preliminary data about PSPA7_1846's potential role.

For a robust experimental design investigating PSPA7_1846 function, consider:

• Controlled comparison groups: wild-type vs. PSPA7_1846 knockout or various expression levels

• Between-subjects or within-subjects design depending on the experimental system

• Multiple measurement methods to validate observations

• Appropriate controls for expression system artifacts

• Sufficient sample sizes determined by power analysis

For example, to study membrane integrity roles, researchers might design an experiment where the independent variable is PSPA7_1846 expression (normal, overexpressed, knocked-down) and the dependent variable is membrane permeability measured through fluorescent dye leakage assays. Multiple control groups and technical replicates would be essential to establish causality between PSPA7_1846 and the observed membrane phenotypes .

What are the key considerations for preserving PSPA7_1846 structure during purification?

Preserving the native structure of PSPA7_1846 during purification requires careful consideration of buffer conditions and handling procedures. As a membrane protein, PSPA7_1846 necessitates specialized approaches to maintain its structural integrity. The protein should be stored in a Tris-based buffer with 50% glycerol to ensure stability during storage at -20°C or -80°C . Researchers should avoid repeated freeze-thaw cycles which can cause protein denaturation and aggregation.

For membrane protein purification, consider the following methodological approaches:

• Membrane extraction: Use mild detergents (DDM, LDAO, or C12E8) at concentrations just above their critical micelle concentration to solubilize PSPA7_1846 while preserving its native conformation.

• Buffer optimization: Include stabilizing agents such as glycerol (10-20%) and reducing agents like DTT or TCEP to prevent oxidation of cysteine residues.

• Purification strategy: Implement a multi-step purification process combining affinity chromatography (utilizing fusion tags), followed by size exclusion chromatography to obtain homogeneous protein preparations.

• Quality assessment: Monitor protein quality throughout purification using techniques such as dynamic light scattering, circular dichroism, and fluorescence spectroscopy to verify proper folding and stability.

For functional studies, reconstitution of PSPA7_1846 into lipid nanodiscs or liposomes with composition similar to Pseudomonas membranes may help maintain native protein conformation and activity .

How can protein-protein interaction studies be designed for PSPA7_1846?

Investigating protein-protein interactions (PPIs) involving PSPA7_1846 requires specialized approaches due to its membrane-embedded nature. A comprehensive strategy would employ multiple complementary techniques to identify and validate interaction partners.

Recommended methodological approaches include:

• Co-immunoprecipitation (Co-IP): Using antibodies against PSPA7_1846 or its fusion tag to pull down protein complexes, followed by mass spectrometry identification of binding partners. This approach can be particularly effective when performed with membrane fractions solubilized with mild detergents, similar to the methodology demonstrated in other membrane protein studies .

• Bimolecular Fluorescence Complementation (BiFC): By fusing complementary fragments of fluorescent proteins to PSPA7_1846 and potential interacting partners, researchers can visualize interactions in living cells through reconstitution of fluorescence signal when the proteins interact.

• Proximity Labeling: Techniques like BioID or APEX2, where PSPA7_1846 is fused to a proximity-dependent labeling enzyme that biotinylates nearby proteins, allowing for subsequent purification and identification.

• Membrane Yeast Two-Hybrid (MYTH): A specialized yeast two-hybrid system designed for membrane proteins that can screen for interactions between PSPA7_1846 and libraries of potential partners.

When analyzing data from these experiments, researchers should create interaction networks and validate key interactions through multiple independent techniques. For example, an interaction identified by Co-IP could be confirmed using BiFC and further characterized by measuring binding kinetics through surface plasmon resonance or microscale thermophoresis .

What structural analysis techniques are most suitable for PSPA7_1846?

Determining the three-dimensional structure of membrane proteins like PSPA7_1846 presents unique challenges due to their hydrophobicity and requirement for a lipid environment. A multi-technique approach is recommended for comprehensive structural characterization:

• Cryo-Electron Microscopy (Cryo-EM): This technique has revolutionized membrane protein structural biology by allowing visualization of proteins in near-native environments without crystallization. For PSPA7_1846, incorporation into nanodiscs or amphipols can maintain structural integrity during Cryo-EM analysis.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For smaller membrane proteins like PSPA7_1846 (109 amino acids), solution NMR using isotopically labeled protein (15N, 13C) reconstituted in detergent micelles can provide valuable structural information and dynamics.

• X-ray Crystallography: Despite challenges, this technique remains powerful if crystals can be obtained. Lipidic cubic phase (LCP) crystallization has proven successful for many membrane proteins and could be applied to PSPA7_1846.

• Computational Approaches: Modern AI-based protein structure prediction tools like AlphaFold2 can provide preliminary structural models of PSPA7_1846, which can guide experimental design and interpretation .

Data integration from multiple techniques is essential. For example, low-resolution Cryo-EM maps combined with computational models and validated by cross-linking mass spectrometry can yield more comprehensive structural insights than any single method alone.

How can researchers overcome expression challenges specific to PSPA7_1846?

Expression of membrane proteins like PSPA7_1846 frequently encounters challenges including toxicity to host cells, aggregation, and poor folding. Several methodological strategies can address these issues:

• Codon optimization: Analyzing the PSPA7_1846 sequence for rare codons in the expression host and optimizing the coding sequence accordingly can significantly improve expression levels. This is particularly important when expressing bacterial proteins in eukaryotic systems or vice versa .

• Induction conditions optimization: A factorial design experiment testing various combinations of:

  • Induction temperature (15°C, 20°C, 25°C, 30°C)

  • Inducer concentration (0.1mM to 1mM IPTG for E. coli)

  • Cell density at induction (OD600 0.4-0.8)

  • Duration of expression (4h to overnight)

• Host strain selection: For E. coli expression, specialized strains like C41(DE3), C43(DE3), or Lemo21(DE3) designed specifically for membrane proteins often yield better results than standard BL21(DE3) .

• Expression construct design: Including fusion partners known to enhance membrane protein expression (MBP, SUMO) and creating multiple constructs with varying N-terminal and C-terminal boundaries can identify optimal expression conditions.

If expression levels remain low, consider switching to a cell-free expression system, which can produce membrane proteins directly in the presence of lipids or detergents, bypassing toxicity issues encountered in living cells .

What are common pitfalls in PSPA7_1846 functional assays and how can they be addressed?

Functional characterization of PSPA7_1846 presents several methodological challenges that researchers should anticipate and address:

• Detergent interference: Many functional assays can be inhibited by detergents used to solubilize membrane proteins. To address this:

  • Use detergents at the lowest effective concentration

  • Consider reconstitution into lipid nanodiscs or proteoliposomes for detergent-free analysis

  • Include appropriate controls to assess detergent effects on assay components

• Protein orientation: In reconstituted systems, membrane proteins can adopt random orientations. Techniques to address this include:

  • Asymmetric reconstitution protocols using charged lipids

  • Engineering epitope tags on specific protein domains for orientation verification

  • Using oriented immobilization on surfaces through site-specific biotinylation

• Aggregation during functional studies: To prevent aggregation:

  • Include stabilizing agents like glycerol or specific lipids

  • Perform size exclusion chromatography immediately before functional assays

  • Monitor protein monodispersity through dynamic light scattering

• Validating physiological relevance: Connect in vitro observations to in vivo function through:

  • Complementation studies in Pseudomonas aeruginosa PSPA7_1846 knockout strains

  • Site-directed mutagenesis of key residues identified in biochemical assays

  • Correlation of in vitro properties with bacterial phenotypes

By anticipating these challenges and implementing appropriate methodological controls, researchers can generate more reliable and reproducible data on PSPA7_1846 function .