Recombinant Rhizobium loti UPF0060 membrane protein mll7841 (mll7841)

What is the basic structure and composition of Recombinant Rhizobium loti UPF0060 membrane protein mll7841?

Recombinant Rhizobium loti UPF0060 membrane protein mll7841 is a full-length protein consisting of 107 amino acids. The complete amino acid sequence is: MTYLFYTAAALAEIAGCFSVWAWWRLERSPLWLAPGFVSLLLFAWLLALVDTNAAGRAYA AYGGIYIAASLAWLWLVEGVRPDRWDLAGAALCIAGASLILLAPRGA. When produced as a recombinant protein, it typically contains an N-terminal His-tag to facilitate purification. The protein is expressed in E. coli expression systems and purified to greater than 90% purity as determined by SDS-PAGE analysis .

How is Recombinant Rhizobium loti UPF0060 membrane protein mll7841 properly stored and reconstituted for experimental use?

Storage Protocol:

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

• After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles

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

• For long-term storage, add 5-50% glycerol (recommended final concentration of 50%) and store at -20°C/-80°C

Reconstitution Protocol:

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

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

• Add glycerol to a final concentration of 5-50% for long-term storage

• Avoid repeated freeze-thaw cycles which can significantly reduce protein activity

What is the UPF0060 protein family and how does mll7841 fit within this classification?

The UPF0060 designation refers to "Uncharacterized Protein Family 0060," a classification for membrane proteins with conserved structural motifs but incompletely characterized functions. Rhizobium loti mll7841 belongs to this family of membrane proteins found across various bacterial species. As a member of this family, mll7841 likely shares structural and possibly functional similarities with other UPF0060 proteins, though its specific role in Rhizobium loti remains to be fully elucidated.

The significance of studying UPF0060 family proteins lies in their potential roles in membrane organization, transport, or signaling pathways that may be essential for bacterial survival or symbiotic relationships. In the case of Rhizobium loti, understanding mll7841 could provide insights into the molecular mechanisms underlying plant-microbe interactions, particularly in nitrogen-fixing root nodule formation .

What are the optimal expression conditions for producing Recombinant Rhizobium loti UPF0060 membrane protein mll7841?

Expression System Selection:
The optimal expression system for mll7841 is E. coli, which has been successfully used to produce the recombinant protein with N-terminal His-tag . For membrane proteins like mll7841, several considerations should be addressed:

Optimized Expression Protocol:

• Vector selection: Use vectors with strong, inducible promoters (e.g., T7) and appropriate tag placement (N-terminal His-tag for mll7841)

• Host strain selection: BL21(DE3) or derivatives optimized for membrane protein expression

• Culture conditions:

  • Initial growth at 37°C to OD600 of 0.6-0.8

  • Temperature reduction to 16-20°C before induction

  • Induction with low IPTG concentration (0.1-0.5 mM)

  • Extended expression time (16-20 hours) at lower temperature

Addressing Common Challenges:

• Hydrophobicity issues: Membrane proteins like mll7841 contain hydrophobic regions that can cause aggregation. Lower expression temperatures and specialized E. coli strains can mitigate this issue .

• Codon optimization: Since Rhizobium loti may have different codon usage than E. coli, codon optimization or use of strains containing rare tRNAs may improve expression .

• Protein toxicity: If mll7841 exhibits toxicity to E. coli, using tightly controlled inducible systems and reduced expression temperatures is recommended .

What purification strategies yield the highest purity and activity for Recombinant Rhizobium loti UPF0060 membrane protein mll7841?

Recommended Purification Workflow:

• Cell Lysis and Membrane Fraction Isolation:

  • Lyse cells using sonication or high-pressure homogenization in buffer containing detergents suitable for membrane protein extraction

  • Separate membrane fraction by ultracentrifugation

  • Solubilize membrane proteins using appropriate detergents (e.g., DDM, LDAO)

• Immobilized Metal Affinity Chromatography (IMAC):

  • Apply solubilized protein to Ni-NTA or similar matrix

  • Wash with increasing imidazole concentrations to remove non-specific binding

  • Elute with buffer containing high imidazole concentration (250-500 mM)

  • For higher purity, use stringent washing with increased imidazole concentrations to distinguish full-length protein from truncated products

• Size Exclusion Chromatography:

  • Further purify by gel filtration to remove aggregates and ensure monodispersity

  • Analyze fractions by SDS-PAGE to confirm >90% purity

• Quality Control:

  • Verify purity using SDS-PAGE

  • Confirm identity using Western blot or mass spectrometry

  • Assess structural integrity using circular dichroism if functional assays are available

How can researchers troubleshoot common issues in recombinant mll7841 protein expression?

Problem: Low Expression Yields

• Solution: Optimize growth conditions (temperature, media composition, induction timing)

• Method: Compare expression at different temperatures (37°C, 30°C, 25°C, 16°C)

• Analysis: Evaluate protein levels in soluble vs. insoluble fractions at each condition

Problem: Formation of Inclusion Bodies

• Solution: Reduce expression rate by lowering temperature and inducer concentration

• Method: Test induction with 0.1 mM IPTG at 16°C for overnight expression

• Alternative approach: Attempt inclusion body solubilization and refolding if native conditions fail

Problem: Truncated Products

• Solution: Use expression vectors with fusion tags at both N and C termini

• Method: Increase imidazole concentration during elution to select for full-length proteins containing both tags

• Verification: Confirm full-length product by Western blot using antibodies against both terminal tags

Problem: Protein Instability

• Solution: Optimize buffer conditions with stabilizing agents

• Method: Test various buffers with additives (glycerol, specific detergents, salt concentrations)

• Storage: Aliquot protein and avoid repeated freeze-thaw cycles

What bioinformatic approaches can predict structural features of mll7841 and inform experimental design?

Primary Sequence Analysis:

• Apply transmembrane prediction algorithms (TMHMM, Phobius) to identify membrane-spanning regions

• The hydrophobic nature of mll7841's sequence (MTYLFYTAAALAEIAGCFSVWAWWRLERSPLWLAPGFVSLLLFAWLLALVDTNAAGRAYA AYGGIYIAASLAWLWLVEGVRPDRWDLAGAALCIAGASLILLAPRGA) suggests multiple transmembrane domains

Structural Homology Modeling:

• Identify structural homologs through sequence alignment with characterized proteins

• Generate 3D models using comparative modeling platforms (SWISS-MODEL, I-TASSER)

• Validate models through Ramachandran plot analysis and QMEAN scoring

Functional Domain Prediction:

• Use InterPro, Pfam databases to identify conserved domains

• Analyze conservation patterns across UPF0060 family members from different species

• Predict potential binding sites or active centers through conservation mapping

Design Implications:

• Target predicted exposed regions for antibody generation

• Design constructs excluding transmembrane regions for soluble domain expression

• Identify potential sites for site-directed mutagenesis based on conserved residues

What experimental techniques are most appropriate for determining the membrane topology of mll7841?

Protease Accessibility Mapping:

• Express mll7841 in membrane vesicles or proteoliposomes

• Treat with membrane-impermeable proteases

• Analyze protected fragments by mass spectrometry

• Map cleavage sites to determine regions exposed on each side of the membrane

Reporter Fusion Assays:

• Generate systematic fusions of mll7841 segments with reporter proteins (GFP, PhoA)

• Express in E. coli and assess reporter activity

• PhoA is active in periplasm, while GFP is fluorescent in cytoplasm

• Complementary signals help map topology accurately

Cysteine Accessibility Methods:

• Replace native cysteines with alanine

• Introduce single cysteines at strategic positions

• Probe accessibility with membrane-permeable and impermeable thiol reagents

• Determine location relative to membrane based on labeling patterns

Cryo-EM or X-ray Crystallography:
For high-resolution structural determination, purify protein in detergent micelles or lipid nanodiscs and apply appropriate structural biology techniques, though these approaches represent significant technical challenges for membrane proteins like mll7841.

How can protein-protein interaction studies reveal functional insights about mll7841 in Rhizobium loti biology?

Pull-down Assays and Co-immunoprecipitation:

• Use His-tagged mll7841 as bait protein with Rhizobium loti lysate

• Identify interacting partners by mass spectrometry

• Validate interactions through reciprocal pull-downs or co-immunoprecipitation

• Apply detergent screens to maintain membrane protein interactions

Bacterial Two-Hybrid Systems:

• Develop constructs with mll7841 fused to split reporter protein domains

• Screen against Rhizobium loti genomic library

• Identify positive interactions through reporter activation

• Verify with orthogonal methods like FRET or BiFC

In vivo Crosslinking:

• Express mll7841 with photo-activatable or chemical crosslinkers

• Induce crosslinking in living bacteria under relevant conditions

• Isolate complexes and identify components by mass spectrometry

• Map interaction sites through crosslink position analysis

Interpretation Framework:

• Compare interacting partners with known symbiosis or membrane organization factors

• Analyze interactions under different physiological conditions (free-living vs. symbiotic)

• Connect findings to Rhizobium-Lotus symbiosis molecular mechanisms

What methods can assess the role of mll7841 in Rhizobium loti symbiotic relationships with Lotus plants?

Gene Knockout and Complementation Studies:

• Generate mll7841 deletion mutants in Rhizobium loti

• Assess mutant phenotypes in:

  • Free-living growth conditions

  • Plant infection efficiency

  • Nodule formation and development

  • Nitrogen fixation capacity

• Complement with wild-type and mutant versions of mll7841

• Quantify restoration of symbiotic phenotypes

Transcriptional Profiling:

• Compare mll7841 expression across developmental stages of symbiosis

• Identify co-regulated genes through RNA-Seq analysis

• Map to known symbiotic pathways and regulons

• Validate expression patterns with qRT-PCR and reporter fusions

Microscopy-Based Approaches:

• Generate fluorescently tagged mll7841 constructs

• Track localization during bacterial infection and nodule development

• Co-localize with known symbiosis markers

• Correlate localization patterns with specific symbiotic stages

Host Genotype Influence Analysis:
This approach would examine how different Lotus genotypes affect mll7841 function, similar to how Rhizobium leguminosarum studies showed host genotype determines infection patterns :

How can advanced microscopy techniques elucidate mll7841 localization and dynamics during symbiosis?

Super-Resolution Microscopy Approaches:

• PALM/STORM for Nanoscale Localization:

  • Tag mll7841 with photoactivatable fluorescent proteins

  • Achieve 20-30 nm resolution to visualize membrane distribution

  • Quantify clustering or dispersion in different symbiotic stages

• Single-Molecule Tracking:

  • Label mll7841 sparsely to track individual molecules

  • Calculate diffusion coefficients in different membrane regions

  • Identify confined regions suggesting functional membrane domains

• FRET-Based Interaction Mapping:

  • Generate donor-acceptor pairs with potential interaction partners

  • Measure FRET efficiency in living bacteria during infection

  • Map interaction networks temporally during symbiosis progression

Sample Preparation Considerations:

• Develop fixation protocols preserving membrane structures

• Optimize infection and nodulation stages for imaging

• Design microfluidic devices for live imaging of infection processes

Data Analysis Framework:

• Apply particle tracking algorithms to quantify movement parameters

• Use cluster analysis to identify membrane domain formation

• Correlate localization patterns with infection efficiency metrics

What comparative genomic approaches can reveal evolutionary insights about mll7841 within rhizobial species?

Phylogenetic Analysis Framework:

• Identify mll7841 homologs across rhizobial and related bacterial species

• Construct phylogenetic trees to map evolutionary relationships

• Calculate selection pressure (dN/dS ratios) on different protein regions

• Correlate conservation patterns with host specificity traits

Synteny Analysis:

• Examine genomic context of mll7841 across species

• Identify conserved gene neighborhoods or operonic structures

• Map rearrangements or horizontal gene transfer events

• Connect genomic organization to functional pathways

Host Range Correlation:

• Compare mll7841 sequence variations between rhizobia with different host specificities

• Identify potential host-specificity determinant regions

• Test chimeric proteins through cross-species complementation

• Establish structure-function relationships for host recognition

Evolutionary Rate Analysis:

• Calculate evolutionary rates across different protein domains

• Identify regions under purifying or diversifying selection

• Connect evolutionary patterns to functional constraints or adaptations

• Develop hypotheses about key functional residues for experimental validation

How can mll7841 research inform broader understanding of membrane protein structure-function relationships?

Comparative Analysis with Other UPF0060 Family Members:

• Align mll7841 with structurally characterized UPF0060 proteins

• Identify conserved structural motifs across bacterial phyla

• Map sequence conservation onto structural models

• Generate hypotheses about functional mechanisms based on conserved features

Integration with Systems Biology Approaches:

• Map mll7841 into protein-protein interaction networks

• Identify functional modules containing mll7841

• Connect to broader cellular pathways through network analysis

• Develop predictive models for membrane protein organization

Translation to Biotechnology Applications:

• Evaluate mll7841 as a potential scaffold for membrane protein engineering

• Assess utility in synthetic biology applications for plant-microbe interactions

• Explore potential as a target for enhancing symbiotic relationships

• Develop methodologies applicable to other challenging membrane proteins

What are methodological considerations for developing antibodies or other detection reagents against mll7841?

Epitope Selection Strategy:

• Analyze predicted topology to identify exposed regions

• Synthesize peptides corresponding to hydrophilic loops

• Use bioinformatic tools to select regions with:

  • High antigenicity scores

  • Low sequence similarity to host proteins

  • Minimal post-translational modification sites

Production Approaches:

• Peptide Antibodies:

  • Generate KLH-conjugated peptides from predicted exposed regions

  • Immunize rabbits or mice using standard protocols

  • Purify antibodies through affinity chromatography

• Recombinant Antibody Fragments:

  • Screen phage display libraries against purified mll7841

  • Select high-affinity binders through iterative panning

  • Express and purify single-chain antibodies or nanobodies

Validation Protocol:

• Test specificity against purified protein by Western blot

• Verify recognition of native protein in membrane fractions

• Confirm specificity through genetic knockouts as negative controls

• Assess cross-reactivity with related proteins from other species

Application-Specific Optimization:

• For immunofluorescence: Optimize fixation to preserve epitope accessibility

• For immunoprecipitation: Test detergent compatibility with epitope recognition

• For ELISA development: Determine optimal coating conditions and detection limits

How can researchers develop functional assays to characterize the biochemical activities of mll7841?

Transport Assay Development:

• Reconstitute purified mll7841 in proteoliposomes

• Load vesicles with fluorescent reporter molecules

• Measure fluorescence changes upon substrate addition

• Characterize transport kinetics and substrate specificity

Protein-Lipid Interaction Assays:

• Employ lipid overlay assays with varied membrane lipids

• Analyze lipid preferences through liposome binding assays

• Study effects of lipid composition on protein activity

• Connect lipid interactions to functional hypotheses

Structural Dynamics Studies:

• Introduce site-specific labels (fluorophores or EPR probes)

• Measure conformational changes under different conditions

• Correlate structural dynamics with functional states

• Develop structural models of the functional cycle

High-Throughput Screening Approaches:

• Design fluorescence-based assays adaptable to multiwell formats

• Screen compound libraries for modulators of mll7841 activity

• Validate hits through secondary assays and structure-activity relationships

• Develop chemical tools for functional analysis in vivo