Recombinant Photorhabdus luminescens subsp. laumondii UPF0208 membrane protein plu3094 (plu3094)

What is plu3094 and what are its basic structural properties?

Plu3094 is a UPF0208 membrane protein from Photorhabdus luminescens subspecies laumondii. It is a 151 amino acid protein with the UniProt ID Q7N2I2. The full amino acid sequence is: MSAIPPTSNGWLRKMQLGQQYMKTWPIEKQLAPMFPENRIIKATRFGIRFMPPLAIFTLTWQIALGGQLGPAVATALFACSLPMQGLWWLGKRASTPLPAALLKWFHEIRDKFAAAGITMAPVQQTPTYQSLAELLKRAFKQLDRSFLDDI . As a membrane protein, it likely contains hydrophobic regions that facilitate its integration into cellular membranes, though specific membrane topology studies are still developing in the literature.

How is recombinant plu3094 typically produced for research purposes?

The recombinant form of plu3094 is typically produced in E. coli expression systems with an N-terminal His-tag for purification purposes . The full-length protein (amino acids 1-151) is expressed and purified using affinity chromatography techniques that exploit the His-tag. After purification, the protein is typically supplied as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . For most research applications, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, with addition of 5-50% glycerol for long-term storage.

How does the membrane topology of plu3094 influence its functional properties?

The membrane topology of plu3094 remains under investigation, but analysis of its amino acid sequence suggests it contains multiple hydrophobic regions characteristic of integral membrane proteins. Researchers studying this protein should consider employing computational prediction tools such as TMHMM, Phobius, or MEMSAT to predict transmembrane domains before designing experiments. Experimental verification of these predictions can be achieved through techniques such as cysteine scanning mutagenesis combined with accessibility studies, or by epitope insertion strategies. Understanding the topology is crucial for structure-function relationship studies, as the orientation of functional domains relative to the membrane can significantly impact interactions with other proteins and substrates.

What are the challenges in crystallizing plu3094 for structural studies?

As with many membrane proteins, crystallizing plu3094 presents significant challenges. Detergent selection is critical, as it must effectively solubilize the protein while maintaining its native conformation. Initial screening should include a panel of detergents such as DDM, LDAO, and OG at various concentrations. The presence of the His-tag may introduce flexibility that hinders crystal formation; therefore, researchers might consider tag removal through proteolytic cleavage or protein engineering approaches. Lipidic cubic phase crystallization has proven successful for many membrane proteins and could be attempted if traditional vapor diffusion methods fail. Thermal stability assays using fluorescent dyes can help identify buffer conditions that enhance protein stability prior to crystallization attempts.

How does the transcriptional regulation of plu3094 vary across different Photorhabdus luminescens phenotypic variants?

Transcriptomic analyses of Photorhabdus luminescens have revealed that phenotypic variants display differential gene expression patterns. In the VAR* variant, which exhibits delayed pathogenicity, significant changes in transcriptional regulation have been observed compared to wild-type strains . Although specific data on plu3094 expression is not directly reported, related membrane proteins show altered expression patterns in these variants. Researchers investigating plu3094 regulation should employ qRT-PCR to quantify expression levels across different variants and growth conditions. RNA-seq analysis can provide a more comprehensive view of how plu3094 expression correlates with other genes in relevant pathways. Promoter-reporter fusion constructs can help identify regulatory elements controlling plu3094 expression.

What are the optimal conditions for reconstituting lyophilized recombinant plu3094?

For optimal reconstitution of lyophilized recombinant plu3094, researchers should first briefly centrifuge the vial before opening to ensure the product is at the bottom of the tube. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being standard for many applications) . The reconstitution process should be gentle to avoid protein denaturation - slow pipetting rather than vortexing is advised. The solution can be allowed to stand at room temperature for 10-15 minutes to ensure complete solubilization before aliquoting. Reconstituted protein should be stored at -20°C/-80°C and repeated freeze-thaw cycles should be strictly avoided .

How can researchers verify the functional integrity of recombinant plu3094 after purification?

Verifying the functional integrity of recombinant plu3094 requires multiple approaches:

Additionally, functional assays should be designed based on hypothesized functions, such as channel activity measurements if ion transport is suspected, or binding assays if receptor functions are predicted.

What are the most effective protein extraction methods for obtaining native plu3094 from Photorhabdus luminescens?

Extracting native plu3094 from Photorhabdus luminescens presents unique challenges due to its membrane localization. A comprehensive approach includes:

• Cell harvesting during exponential growth phase when membrane protein expression is often optimal

• Cell lysis using techniques that preserve membrane protein structure:

  • Gentle mechanical disruption with glass beads

  • Enzymatic treatment with lysozyme followed by osmotic shock

  • French press at moderate pressure (15,000-20,000 psi)

• Membrane fraction isolation through differential centrifugation:

  • Low-speed centrifugation (5,000 × g) to remove intact cells and debris

  • Ultracentrifugation (100,000 × g) to collect membrane fractions

• Solubilization screening with different detergents:

  • Non-ionic detergents (DDM, Triton X-100) at concentrations just above CMC

  • Zwitterionic detergents (LDAO, CHAPS) for situations requiring higher solubilization efficiency

• Purification using a combination of:

  • Affinity chromatography (if antibodies against plu3094 are available)

  • Ion exchange chromatography

  • Size exclusion chromatography as a final polishing step

Western blotting should be performed throughout the process to track protein recovery and purity.

How can plu3094 be utilized in studying bacterial adaptation mechanisms?

Recombinant plu3094 can serve as a valuable tool for investigating bacterial adaptation mechanisms in Photorhabdus luminescens. Researchers can explore its potential roles in:

• Membrane remodeling during host adaptation by comparing expression levels between nematode-associated and insect-infecting phases

• Stress response pathways by examining interactions with known stress response proteins

• Signaling networks through identification of interaction partners using proximity labeling approaches such as BioID or APEX

• Environmental sensing by testing functional changes under various conditions mimicking host environments

A targeted approach would involve creating knockout or knockdown strains to observe phenotypic changes in different environments. Complementation studies using the recombinant protein can confirm observed phenotypes are specifically related to plu3094 function. Localization studies using fluorescent fusion proteins can reveal dynamic changes in protein distribution during adaptation processes.

What insights can comparative studies between plu3094 and related UPF0208 family proteins provide?

Comparative studies between plu3094 and other UPF0208 family members can yield valuable insights into evolutionary conservation and functional specialization. Research approaches should include:

• Phylogenetic analysis to establish evolutionary relationships between UPF0208 proteins across bacterial species

• Structural comparison through homology modeling based on any available crystal structures of family members

• Functional complementation experiments to determine if UPF0208 proteins from other species can rescue plu3094 knockout phenotypes

• Domain swapping experiments to identify functionally critical regions

• Binding partner identification through co-immunoprecipitation followed by mass spectrometry to compare interactomes

These comparative approaches can reveal conserved functional motifs and species-specific adaptations. Understanding the differences between UPF0208 proteins in mutualistic versus pathogenic bacteria may provide insights into their role in host-microbe interactions and virulence mechanisms.

How does the expression of plu3094 correlate with virulence mechanisms in Photorhabdus luminescens?

Investigating the relationship between plu3094 expression and virulence mechanisms requires integrated experimental approaches:

• Quantitative expression analysis across different growth phases and infection stages using qRT-PCR

• Correlation analysis with known virulence factors, particularly examining co-expression patterns with insecticidal toxins like Pit and PirB

• Response to host-derived signals by exposing bacteria to insect hemolymph components and measuring expression changes

• Virulence assessment using:

  • Knockout strains in insect infection models

  • Overexpression strains to identify potential gain-of-function phenotypes

  • Complementation with site-directed mutants to identify critical functional residues

Previous research on P. luminescens variants has demonstrated that transcriptomic changes are associated with altered pathogenicity, particularly in oxidative stress response genes and toxin production . Similar mechanisms might apply to plu3094, particularly if it functions in membrane protection against host immune responses or environmental stresses encountered during infection.

What are the optimal storage conditions to maintain stability of recombinant plu3094?

Maintaining stability of recombinant plu3094 requires careful attention to storage conditions. The lyophilized protein should be stored at -20°C/-80°C upon receipt . After reconstitution, the protein should be aliquoted to avoid repeated freeze-thaw cycles. For short-term use, working aliquots can be stored at 4°C for up to one week . The storage buffer containing Tris/PBS with 6% trehalose at pH 8.0 helps maintain stability, and addition of 5-50% glycerol to reconstituted protein is recommended for long-term storage . Researchers should monitor protein stability over time using analytical techniques such as SDS-PAGE and activity assays to establish site-specific optimal storage conditions.

How can researchers troubleshoot poor expression or solubility issues with recombinant plu3094?

When encountering expression or solubility issues with recombinant plu3094, researchers should implement a systematic troubleshooting approach:

Additionally, fusion partners like MBP or SUMO can enhance solubility, while specialized E. coli strains (C41/C43, Rosetta, etc.) may improve membrane protein expression. Chaperone co-expression strategies can also facilitate proper folding of challenging membrane proteins.