Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni 50S ribosomal protein L35 (rpmI)

What is the basic structure and function of the 50S ribosomal protein L35 in Leptospira interrogans?

The 50S ribosomal protein L35 (rpmI) in L. interrogans is a small ribosomal protein comprising 67 amino acids with the sequence MPKLKTNRAA AKRFKFTKNN NIKRKSMNTR HILTKKGPKR RRRLRGLTLV HNSDWKSIVR LMPYGVR . As a component of the large ribosomal subunit, L35 contributes to protein synthesis machinery essential for bacterial survival. The protein contains characteristic ribosomal protein folding patterns that facilitate RNA-protein interactions within the ribosome structure.

Structural features:

• Full-length protein spanning amino acids 1-67

• Rich in basic amino acids (K, R) that facilitate interaction with ribosomal RNA

• Contains structural motifs typical of ribosomal proteins that maintain tertiary structure

The recombinant form is typically expressed with a histidine tag to facilitate purification, with verification by SDS-PAGE showing >85% purity .

How does L35 protein expression differ between virulent and avirulent Leptospira strains?

Expression patterns of ribosomal proteins, including L35, can vary between virulent and avirulent strains, though this specific protein has not been extensively characterized in this context. Research on other leptospiral proteins shows significant differences in expression between virulent strains like L. interrogans Fiocruz L1-130 (low-passage) and culture-attenuated strains like M20 (high-passage) .

When examining leptospiral protein expression:

• Virulent strains often maintain expression profiles optimized for host infection

• Culture-attenuated strains may show altered expression patterns due to adaptation to laboratory growth conditions

• Expression differences can be detected using western blotting with protein-specific antibodies

For accurate assessment of L35 expression differences, quantitative western blotting comparing whole-cell lysates from virulent L. interrogans strain L1-130 and attenuated strain M20 would be recommended, similar to methodologies used for other leptospiral proteins .

What are the optimal conditions for expressing and purifying recombinant L. interrogans 50S ribosomal protein L35?

Optimal expression and purification of recombinant L35 requires careful consideration of expression systems, growth conditions, and purification strategies.

Expression system recommendations:

• Baculovirus expression systems have been successfully employed for recombinant leptospiral proteins

• E. coli systems may be used with codon optimization for leptospiral genes

Purification protocol:

• Express protein with histidine tag for affinity purification

• Harvest cells by centrifugation (3,000 × g, 10 min)

• Resuspend in appropriate buffer and lyse cells

• Purify using nickel affinity chromatography

• Verify purity by SDS-PAGE (target >85%)

• Consider additional purification steps if higher purity is required

Quality control measures:

• SDS-PAGE analysis to confirm expected molecular weight and purity

• Western blotting with anti-histidine antibodies to verify identity

• Mass spectrometry for sequence confirmation

What storage and handling protocols maximize stability of recombinant L35 protein?

Proper storage and handling are critical for maintaining recombinant L35 protein stability and activity.

Storage recommendations:

• Avoid repeated freeze-thaw cycles which significantly reduce protein stability

• Store working aliquots at 4°C for up to one week

• For long-term storage, maintain at -20°C/-80°C

• Lyophilized form maintains stability for approximately 12 months at -20°C/-80°C

• Liquid form typically maintains stability for 6 months at -20°C/-80°C

Reconstitution protocol:

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

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

• Add glycerol to 5-50% (recommended final concentration: 50%) for long-term storage

• Aliquot to minimize freeze-thaw cycles

How can cross-reactivity issues be mitigated when developing antibodies against L35 protein?

Antibody cross-reactivity is a significant challenge in Leptospira research. Studies with other leptospiral proteins have shown that polyclonal antibodies often cross-react with structurally similar proteins .

Strategies to minimize cross-reactivity:

• Use highly purified recombinant protein for immunization

• Consider monoclonal antibody development instead of polyclonal antibodies

• Select unique peptide regions for antibody production

• Validate antibody specificity using multiple techniques:

  • Western blotting against recombinant protein and whole cell lysates

  • ELISA with related and unrelated proteins

  • Immunofluorescence microscopy with various Leptospira strains

Cross-reactivity assessment:

Research on leptospiral LRR-proteins demonstrated that polyclonal antibodies can recognize multiple proteins sharing similar domains, necessitating careful validation .

How can recombinant L35 protein be adapted for use in leptospirosis diagnostic assays?

While L35 protein has not been specifically evaluated for diagnostic applications, research on other recombinant leptospiral proteins provides a framework for developing such assays.

Diagnostic assay development considerations:

• Assess immunoreactivity with patient sera in ELISA format

• Determine sensitivity and specificity thresholds

• Compare performance against established diagnostic antigens

Studies with recombinant leptospiral proteins like LipL32 showed promising diagnostic utility, with IgG ELISAs demonstrating 56% sensitivity in acute phase and 94% in convalescent phase of leptospirosis . Similar evaluation protocols could be applied to recombinant L35:

Evaluation protocol:

• Establish cutoff values using sera from healthy individuals in endemic regions (targeting 96% specificity)

• Test paired sera from confirmed leptospirosis cases

• Assess cross-reactivity with sera from patients with clinically similar diseases (dengue, hepatitis, etc.)

• Calculate sensitivity and specificity metrics

The potential advantages of ribosomal proteins for diagnostics include their conserved nature and potential immunogenicity during infection.

What techniques are most effective for studying interactions between L35 and other ribosomal components?

Understanding L35 interactions with other ribosomal components requires specialized techniques for protein-protein and protein-RNA interactions.

Recommended methodologies:

• Pull-down assays:

  • Express recombinant L35 with affinity tag

  • Incubate with leptospiral lysate or purified ribosomal fractions

  • Analyze co-precipitated proteins by mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize recombinant L35 on sensor chip

  • Flow potential binding partners over surface

  • Measure binding kinetics and affinity constants

• Structural biology approaches:

  • X-ray crystallography of L35 alone or in complex

  • Cryo-electron microscopy of ribosomal assemblies

  • Nuclear magnetic resonance for smaller interaction domains

• Crosslinking mass spectrometry:

  • Use chemical crosslinkers to capture transient interactions

  • Identify interaction sites through mass spectrometry analysis

These techniques would follow similar protocols to those used for studying other leptospiral proteins, adapting methods used for LRR-proteins that were shown to interact with host components .

How does L35 protein from pathogenic L. interrogans compare to homologous proteins in saprophytic Leptospira species?

Comparison of proteins between pathogenic and saprophytic Leptospira species provides insights into evolutionary adaptations and potential virulence contributions.

Comparative analysis approaches:

• Sequence alignment to identify conserved and variable regions

• Expression pattern analysis in different species

• Structural modeling to predict functional differences

Research on other leptospiral proteins has shown significant differences between pathogenic and saprophytic species. For example, studies with LRR-proteins demonstrated that LIC11051 was detected in pathogenic L. interrogans but absent in saprophytic L. biflexa, while LIC11505 was present in both but with lower intensity in L. biflexa .

Similar methodology could be applied to L35 protein:

• Western blotting of whole-cell lysates from multiple Leptospira species

• Quantitative PCR to measure expression levels

• Immunofluorescence microscopy to determine localization differences

What are the unexplored potential roles of L35 protein in leptospiral pathogenesis?

Ribosomal proteins can have non-canonical functions beyond protein synthesis, potentially contributing to bacterial pathogenesis. Several research directions could explore these possibilities for L35:

• Investigation of moonlighting functions:

  • Surface exposure and potential host interactions

  • Role in stress response during infection

  • Contribution to antibiotic resistance mechanisms

• Host immune response studies:

  • Recognition by pattern recognition receptors

  • Ability to modulate host inflammatory responses

  • Potential to prevent macrophage cell death similar to other leptospiral components

• Regulatory roles:

  • Potential function in regulating gene expression during different growth phases

  • Adaptation to environmental stresses during host colonization

Research has shown that L. interrogans can prevent macrophage cell death and modulate immune responses , and ribosomal proteins could contribute to these processes if they have functions beyond the ribosome.

How might targeted modifications of recombinant L35 enhance its utility for research applications?

Strategic modifications to recombinant L35 could expand its research applications and improve its utility as a tool for studying leptospiral biology.

Potential modifications:

• Affinity tag optimization:

  • Testing various tag positions (N-terminal vs. C-terminal)

  • Evaluating different tag types for improved solubility or detection

• Fluorescent protein fusions:

  • Creating GFP-L35 fusions for localization studies

  • Developing FRET pairs with other ribosomal proteins

• Functional domain mapping:

  • Creating truncated variants to identify critical regions

  • Site-directed mutagenesis of conserved residues

• Stability enhancements:

  • Engineering for improved thermostability

  • Modifications to reduce aggregation tendency

These approaches would build upon established techniques for recombinant protein engineering and could significantly enhance the utility of L35 as a research tool in leptospirosis studies.