Recombinant Photorhabdus luminescens subsp. laumondii UPF0266 membrane protein plu2700 (plu2700)

What is the Photorhabdus luminescens UPF0266 membrane protein plu2700?

The UPF0266 membrane protein plu2700 is a 152-amino acid transmembrane protein found in Photorhabdus luminescens subsp. laumondii (strain TT01), a gram-negative, bioluminescent bacterium belonging to the Enterobacteriaceae family. The protein is classified as part of the UPF (Uncharacterized Protein Family) 0266, suggesting its function has not been fully characterized. Its amino acid sequence is: MNLNDIALTGLIVLMLAFAVYDEFVVNFLKGKTHLQIKLKRKHKIDALIFIILILIVVYNNITVYGSRLTTYLLLFTILVTIYIAYIRSPKLFFKNNGFFYANTFISYSRIKTMNLSEDGILVIGLENKKLYISVSQIDDLERIYKFLIENR

What is the predicted membrane topology of plu2700?

Based on hydropathy analysis and structural prediction algorithms, plu2700 is predicted to contain multiple transmembrane helices. The hydrophobic regions (particularly residues 9-29 and 45-65) likely span the membrane, while the charged residues (particularly the lysine-rich region around positions 30-40) are likely exposed to the cytoplasmic or periplasmic space. The protein contains a relatively high proportion of hydrophobic amino acids (approximately 60%), consistent with its membrane localization.

What is known about the expression patterns of plu2700 in P. luminescens?

While specific expression data for plu2700 is limited, many membrane proteins in P. luminescens show differential expression patterns during the bacterium's lifecycle phases. The gene may be regulated similarly to other membrane proteins in this organism, potentially through ner gene-mediated regulation, which has been shown to affect primary-form-specific phenotypes in P. luminescens . Northern blot analysis techniques similar to those used for the ner gene could be applied to study plu2700 expression patterns across different growth phases.

What expression systems are most effective for producing recombinant plu2700?

The recombinant plu2700 protein has been successfully expressed in E. coli expression systems with N-terminal His-tagging . Based on available data, the following expression systems have been documented:

For membrane proteins like plu2700, E. coli expression systems typically provide good yields, though proper folding may require optimization of expression conditions.

What are the recommended protocols for purification of recombinant plu2700?

Purification of recombinant plu2700 typically follows standard membrane protein purification procedures:

• Cell Lysis and Membrane Isolation:

  • Harvest cells by centrifugation (5,000×g, 15 min, 4°C)

  • Resuspend in lysis buffer containing protease inhibitors

  • Disrupt cells via sonication or French press

  • Remove cell debris by centrifugation (10,000×g, 20 min, 4°C)

  • Isolate membranes by ultracentrifugation (100,000×g, 1 hour, 4°C)

• Solubilization:

  • Resuspend membrane fraction in solubilization buffer containing appropriate detergents (e.g., DDM, LDAO, or Triton X-100)

  • Incubate with gentle agitation (2-4 hours, 4°C)

  • Remove insoluble material by ultracentrifugation (100,000×g, 30 min, 4°C)

• Affinity Chromatography:

  • Apply solubilized fraction to Ni-NTA or similar affinity resin

  • Wash with buffer containing low imidazole concentrations

  • Elute protein with buffer containing high imidazole concentrations

• Size Exclusion Chromatography:

  • Further purify by gel filtration to remove aggregates and contaminants

  • Analyze purity by SDS-PAGE (expected purity >90%)

What are the optimal storage conditions for maintaining plu2700 stability?

Based on product information, the following storage conditions are recommended for maintaining stability of recombinant plu2700 :

• Storage buffer: Tris-based buffer containing 50% glycerol (or alternatively Tris/PBS-based buffer with 6% trehalose, pH 8.0)

• Short-term storage: 4°C for up to one week

• Long-term storage: -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• For extended storage periods, aliquoting is necessary

When reconstituting lyophilized protein:

• Centrifuge vial briefly before opening

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

• Add glycerol to 5-50% final concentration for storage

What methods are recommended for determining the 3D structure of plu2700?

Several complementary approaches are recommended for structural determination of membrane proteins like plu2700:

How can researchers investigate potential binding partners of plu2700?

Several approaches can be employed to identify potential binding partners:

• Pull-down Assays:

  • Use purified His-tagged plu2700 as bait

  • Incubate with P. luminescens cell lysate

  • Analyze bound proteins by mass spectrometry

• Bacterial Two-Hybrid System:

  • Generate fusion constructs with plu2700

  • Screen against genomic library of P. luminescens

  • Verify interactions with co-immunoprecipitation

• Cross-linking Mass Spectrometry:

  • Apply chemical cross-linkers to stabilize transient interactions

  • Digest and analyze by LC-MS/MS

  • Identify cross-linked peptides to map interaction interfaces

• Co-purification Studies:

  • Analyze proteins that co-purify with tagged plu2700

  • Verify specific interactions with competition assays

What bioinformatic approaches can predict the function of plu2700?

Given the limited functional characterization of UPF0266 family proteins, bioinformatic approaches can provide valuable insights:

• Sequence Homology Analysis:

  • BLAST against characterized proteins

  • Multiple sequence alignment with other UPF0266 family members

  • Conservation analysis across bacterial species

• Protein Domain Prediction:

  • Search for conserved domains using Pfam, SMART, or InterPro

  • Identify functional motifs that may suggest activity

• Structural Homology Modeling:

  • Generate 3D models using AlphaFold2 or RoseTTAFold

  • Compare predicted structures with characterized proteins

  • Identify potential binding pockets or catalytic sites

• Genomic Context Analysis:

  • Examine neighboring genes in the P. luminescens genome

  • Identify potential operons or functionally related gene clusters

  • Compare with syntenic regions in related organisms

What approaches can determine if plu2700 is involved in bacterial virulence?

Several experimental approaches can elucidate potential roles in virulence:

• Gene Knockout/Knockdown Studies:

  • Generate plu2700 deletion mutants using CRISPR-Cas9 or homologous recombination

  • Compare virulence between wild-type and mutant strains in insect models

  • Complement mutants to confirm phenotype specificity

• Transcriptional Analysis:

  • Compare plu2700 expression during different phases of infection

  • Use qRT-PCR or RNA-Seq to quantify expression changes

  • Correlate with expression of known virulence factors

• Host-Pathogen Interaction Assays:

  • Investigate interactions with host cell components

  • Test effects on host immune responses

  • Evaluate contribution to bacterial survival in host

• Toxin Secretion Analysis:

  • Determine if plu2700 affects secretion of known toxins like Tc toxins, Pir proteins, or Mcf toxins

  • Analyze secretome profiles of wild-type versus mutant strains

  • Test for altered cytotoxicity in insect cell cultures

How can researchers investigate the role of plu2700 in membrane integrity or transport?

As a membrane protein, plu2700 may function in membrane integrity or transport processes:

• Membrane Permeability Assays:

  • Compare membrane integrity in wild-type versus plu2700 mutants

  • Use fluorescent dyes (e.g., propidium iodide) to assess permeability

  • Measure susceptibility to osmotic stress or membrane-targeting antibiotics

• Transport Assays:

  • Reconstitute purified protein into liposomes

  • Test for transport of various substrates (ions, small molecules)

  • Use fluorescent indicators or radiolabeled compounds to track transport

• Electrophysiology:

  • Incorporate protein into planar lipid bilayers

  • Measure conductance changes in response to potential substrates

  • Characterize channel properties if applicable

• Localization Studies:

  • Generate fluorescently tagged versions of plu2700

  • Determine subcellular localization within bacterial cells

  • Investigate potential co-localization with known transport systems

How can researchers study potential roles of plu2700 in bacterial-nematode symbiosis?

P. luminescens forms a symbiotic relationship with nematodes, which might involve membrane proteins like plu2700:

• Colonization Assays:

  • Compare ability of wild-type versus plu2700 mutants to colonize nematode hosts

  • Quantify bacterial loads in nematodes using CFU counts or qPCR

  • Assess nematode development and reproduction when colonized by mutants

• Transcriptional Profiling:

  • Compare plu2700 expression during free-living versus symbiotic phases

  • Identify co-regulated genes that may function in symbiosis

  • Use RNA-Seq to capture global transcriptional changes

• In vivo Imaging:

  • Generate fluorescently labeled bacterial strains

  • Track colonization patterns in nematode hosts

  • Compare localization of wild-type versus mutant bacteria

• Metabolite Exchange Studies:

  • Investigate whether plu2700 affects nutrient exchange between bacteria and nematodes

  • Analyze metabolomic profiles using LC-MS

  • Identify altered metabolic pathways

What advanced techniques can be used to study protein-lipid interactions for plu2700?

Membrane proteins often have specific lipid requirements for function:

• Lipid Nanodisc Reconstitution:

  • Incorporate purified plu2700 into nanodiscs with defined lipid composition

  • Test functional activity in different lipid environments

  • Analyze protein stability and conformation changes

• Native Mass Spectrometry:

  • Analyze protein-lipid complexes directly

  • Identify specifically bound lipids that co-purify with the protein

  • Determine binding affinities for different lipids

• Molecular Dynamics Simulations:

  • Model protein behavior in various membrane environments

  • Predict lipid binding sites and interaction energies

  • Simulate conformational changes in response to lipid binding

• Fluorescence-based Techniques:

  • Employ FRET to measure protein-lipid interactions

  • Use environment-sensitive fluorescent probes to detect conformational changes

  • Monitor lipid-dependent changes in protein dynamics

How does plu2700 compare to other UPF0266 family proteins across bacterial species?

Comparative analysis can provide functional insights:

Comparative genomic analysis reveals that UPF0266 family proteins are conserved across Enterobacteriaceae, suggesting an important cellular function. The highest conservation is observed in the predicted transmembrane regions, while the hydrophilic loops show greater sequence variation. This pattern suggests that the membrane-spanning regions may be critical for structural integrity or core function, while the variable regions might confer species-specific roles.

What are common challenges when working with recombinant plu2700 and how can they be addressed?

Membrane proteins present specific challenges:

• Low Expression Yields:

  • Optimize expression conditions (temperature, induction time, inducer concentration)

  • Test different E. coli strains (C41(DE3), C43(DE3), Lemo21)

  • Consider codon optimization for heterologous expression

  • Use fusion partners (MBP, SUMO) to improve solubility

• Protein Aggregation:

  • Screen multiple detergents for solubilization (DDM, LDAO, LMNG)

  • Add stabilizing agents (glycerol, specific lipids)

  • Optimize buffer conditions (pH, salt concentration)

  • Consider nanodiscs or amphipols for stabilization

• Poor Purity:

  • Implement multi-step purification strategies

  • Use size exclusion chromatography as a final polishing step

  • Consider additional affinity tags or purification steps

  • Optimize washing conditions during affinity chromatography

• Loss of Activity:

  • Minimize time between purification and functional assays

  • Store protein with appropriate stabilizers

  • Reconstitute into lipid environments that support activity

  • Consider detergent exchange during purification

How can researchers determine if recombinant plu2700 is properly folded?

Assessing proper folding is critical for functional studies:

• Circular Dichroism (CD) Spectroscopy:

  • Analyze secondary structure content

  • Compare with predicted secondary structure

  • Monitor thermal stability and unfolding

• Intrinsic Fluorescence:

  • Measure tryptophan/tyrosine fluorescence

  • Monitor changes under different conditions

  • Assess accessibility of aromatic residues

• Size Exclusion Chromatography:

  • Analyze elution profile

  • Monodisperse peak suggests proper folding

  • Multiple peaks or void volume elution indicates aggregation

• Limited Proteolysis:

  • Well-folded proteins show resistance to proteolysis

  • Compare digestion patterns under native versus denaturing conditions

  • Identify stable domains

What controls should be included in functional assays for plu2700?

Robust experimental design requires appropriate controls:

• Negative Controls:

  • Empty vector-transformed cells

  • Heat-denatured protein

  • Detergent-only samples for membrane assays

  • Buffer-only controls for all experiments

• Positive Controls:

  • Well-characterized membrane proteins of similar size

  • Known channel or transporter proteins in functional assays

  • Proteins with established lipid interactions

• Validation Controls:

  • Complementation with wild-type protein in knockout studies

  • Site-directed mutants targeting predicted functional residues

  • Concentration gradients to establish dose-dependency

• Technical Controls:

  • Biological replicates (minimum n=3)

  • Technical replicates for each biological sample

  • Multiple protein preparations to ensure reproducibility

What is the potential significance of plu2700 in understanding bacterial-host interactions?

While the specific function of plu2700 remains unknown, membrane proteins in pathogenic bacteria often play crucial roles in host interactions:

• Potential Roles in Pathogenesis:

  • May participate in adhesion to host cells

  • Could be involved in sensing host environments

  • Might contribute to evasion of host immune responses

  • Potentially involved in toxin secretion systems

• Evolutionary Significance:

  • Comparative analysis across Photorhabdus species may reveal adaptation signatures

  • Analysis of selection pressure on different protein domains could identify host-interacting regions

  • Conservation patterns may indicate essential functions

• Therapeutic Potential:

  • If involved in virulence, could represent a novel target for anti-infective strategies

  • Structure determination could facilitate rational drug design

  • Understanding membrane protein function contributes to bacterial physiology knowledge

How might CRISPR-Cas9 approaches be applied to study plu2700 function?

CRISPR-Cas9 technology offers powerful tools for functional genomics:

• Gene Knockout Studies:

  • Design sgRNAs targeting plu2700

  • Create clean deletions or insertional inactivation

  • Characterize resulting phenotypes comprehensively

• CRISPRi for Conditional Knockdown:

  • Use catalytically dead Cas9 (dCas9) to repress gene expression

  • Apply inducible systems for temporal control

  • Tune expression levels with different promoters

• CRISPRa for Overexpression:

  • Use dCas9 fusion activators to increase expression

  • Study effects of plu2700 overexpression on bacterial physiology

  • Identify potential dosage-sensitive interactions

• Base Editing Approaches:

  • Introduce specific amino acid changes without double-strand breaks

  • Target conserved residues to assess functional importance

  • Create libraries of variants for high-throughput functional screening

What emerging technologies might advance understanding of plu2700 function?

Several cutting-edge approaches show promise:

• Cryo-Electron Tomography:

  • Visualize protein complexes in their native membrane environment

  • Determine localization patterns within bacterial cells

  • Identify potential interaction partners in situ

• Single-Molecule Techniques:

  • Track individual protein molecules in live cells

  • Measure diffusion dynamics in membranes

  • Detect conformational changes in real-time

• Artificial Intelligence Approaches:

  • Apply machine learning to predict protein function from sequence

  • Use AlphaFold2 or RoseTTAFold for accurate structure prediction

  • Employ deep learning to identify functional motifs

• High-Throughput Screening:

  • Develop reporter systems to monitor plu2700 activity

  • Screen compound libraries for modulators

  • Apply functional genetic screens to identify genetic interactions