Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Probable GTP-binding protein EngB (engB)

What expression systems are most effective for recombinant Leptospira interrogans proteins?

The Escherichia coli Gateway cloning and expression system has proven effective for recombinant Leptospira protein expression. Specifically, the pENTR cloning vector followed by the pDEST17 expression vector allows for the production of recombinant proteins with a six-His tag at the N-terminus, facilitating subsequent purification steps . This system has been successfully employed for multiple Leptospira proteins, including those encoded by LIC10765 and LIC13131, where genes without signal peptides were amplified from the genomic DNA of L. interrogans serovar Copenhageni using specific primers . When designing your expression strategy, consider removing signal peptides to improve solubility while maintaining the functional domains of interest.

For optimal expression of leptospiral proteins, gene sequences should be codon-optimized for E. coli usage. Expression conditions typically require optimization of temperature (often reduced to 18-30°C), IPTG concentration, and induction time to maximize soluble protein yield and minimize inclusion body formation.

What purification techniques yield the highest purity for recombinant Leptospira proteins?

Affinity chromatography using Ni²⁺-charged resin is the most commonly employed primary purification method for His-tagged recombinant leptospiral proteins. This approach allows proteins to be purified from the soluble bacterial fraction with reasonably high efficiency . To achieve research-grade purity, a multi-step purification strategy is recommended:

• Initial capture using immobilized metal affinity chromatography (IMAC)

• Intermediate purification via ion exchange chromatography to remove bacterial contaminants

• Final polishing step using size exclusion chromatography

For proteins that form inclusion bodies, solubilization protocols using 8M urea or guanidine hydrochloride followed by on-column refolding during purification have proven successful for some leptospiral proteins. Protein purity should be verified through SDS-PAGE analysis and, when applicable, mass spectrometry to confirm protein identity and integrity.

How can researchers confirm the proper folding and functionality of recombinant leptospiral proteins?

Verification of proper folding and functional activity requires multiple complementary approaches:

• Circular dichroism (CD) spectroscopy to assess secondary structure composition

• Thermal shift assays to evaluate protein stability

• Functional binding assays to confirm interaction with known ligands

For Leptospira proteins with binding capabilities, such as those containing leucine-rich repeat (LRR) domains, ELISA-based assays can demonstrate specific interactions with host molecules like extracellular matrix components. For example, recombinant LIC11505 shows specific, dose-dependent, and saturable binding to GAGs and integrin receptors, which can be used as positive controls when setting up binding assays for other leptospiral proteins .

What bioinformatic approaches are most useful for predicting the function and structure of uncharacterized Leptospira interrogans proteins?

A comprehensive bioinformatic analysis should incorporate multiple complementary approaches:

• Domain prediction using SMART (http://smart.embl-heidelberg.de) to identify functional domains such as LRR regions, which have been successfully applied to characterize 21 LRR proteins in L. interrogans

• Virulence factor prediction using specialized databases such as VFDB and Victors, which have identified approximately 161-166 genes in L. interrogans strain HP358 that share orthologs with virulence factors in other bacterial species

• Protein localization prediction using algorithms that analyze signal peptides, transmembrane domains, and subcellular targeting signals

For structural predictions, modern tools implementing deep learning approaches like AlphaFold2 can generate high-confidence structural models, particularly useful for proteins with known domains. These predictions should be validated experimentally using techniques such as X-ray crystallography or cryo-electron microscopy when possible.

How can researchers differentiate between membrane-associated and secreted forms of Leptospira proteins in experimental systems?

Distinguishing between membrane-associated and secreted protein forms requires a systematic fractionation approach:

• Collect culture supernatant for secreted protein analysis

• Separate membrane fractions using differential detergent solubilization:

  • Triton X-114 phase partitioning to separate hydrophobic membrane proteins from hydrophilic proteins

  • SDS-extraction for tightly associated membrane proteins

This approach has been successfully applied to characterize LRR proteins like LIC11051 and LIC11505. LIC11051 was found primarily in the secreted fraction, while LIC11505 was detected in both secreted and SDS-soluble fractions .

Quantitative proteomics using methods like iBAQ (intensity-based absolute quantification) can provide relative abundance measurements. For example, in the Triton X-114 fraction, leptospiral LRR proteins were found at concentrations ranging from 2.4 to 52.8 iBAQ, compared to the highly abundant LipL32 at 6388.2 iBAQ .

What experimental approaches best demonstrate the reassociation of secreted Leptospira proteins with the bacterial surface?

To demonstrate protein reassociation with the bacterial surface, researchers should employ a multi-step experimental approach:

• Express and purify the recombinant protein of interest

• Generate specific antibodies against the purified protein

• Incubate intact Leptospira cells with various concentrations of the purified protein

• Detect surface-bound protein using immunofluorescence microscopy or flow cytometry

This methodology has successfully demonstrated that recombinant LIC11505 can specifically bind to intact L. interrogans cells in a dose-dependent manner, suggesting that the secreted native protein could reassociate with the Leptospira surface . This feature has also been hypothesized for other LRR proteins like LIC10831 .

To quantify the binding, researchers should include appropriate controls for non-specific binding and perform dose-response experiments to demonstrate specificity and saturation kinetics.

What methodologies are most appropriate for identifying host cell receptors that interact with leptospiral proteins?

Identification of host receptors requires a systematic approach:

• Initial screening using protein-protein interaction assays:

  • Yeast two-hybrid screening against cDNA libraries from target host tissues

  • Pull-down assays coupled with mass spectrometry

  • Protein microarrays containing host receptor candidates

• Confirmation of specific interactions:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Co-immunoprecipitation from host cell lysates

  • Mammalian cell binding assays with specific receptor blocking

For leptospiral proteins known to interact with extracellular matrix components, systematic screening against purified ECM components has been successful. LRR-containing proteins have demonstrated binding to various host components, including GAGs and integrin receptors . For GTP-binding proteins like EngB, interactions with host cytoskeletal components or signaling proteins would be potential targets for investigation.

How can researchers determine if a recombinant leptospiral protein is immunogenic during natural infection?

Assessment of immunogenicity during natural infection requires:

• Collection of serum samples from:

  • Patients in early and convalescent phases of leptospirosis

  • Healthy individuals as negative controls

  • Patients with other febrile diseases to assess cross-reactivity

• Evaluation of antibody responses using:

  • ELISA to detect IgG and IgM antibodies against the recombinant protein

  • Western blot analysis to confirm specificity

  • IgG subclass determination to characterize the type of immune response

This approach has been successfully applied to recombinant proteins MPL17 and MPL21 (encoded by LIC10765 and LIC13131), demonstrating their reactivity with sera from leptospirosis patients while showing low levels of reactivity with serum samples from healthy individuals . The results provide evidence for in vivo expression during infection and potential utility as diagnostic markers or vaccine candidates.

What experimental design best demonstrates the role of a leptospiral protein in virulence?

A comprehensive experimental approach to determine virulence contributions includes:

• Gene knockout or knockdown studies using techniques optimized for Leptospira

• Complementation studies to confirm phenotype restoration

• Animal infection models comparing wild-type and mutant strains:

  • Hamster acute infection model

  • Mouse colonization model

  • Cell culture invasion and cytotoxicity assays

• Evaluation of bacterial load, dissemination, and host immune responses

For proteins like LRR-containing molecules that may participate in host adhesion, additional assays should assess:

• Bacterial attachment to host cells or tissues

• Resistance to serum killing

• Evasion of innate immune responses

The virulence contribution of proteins can also be inferred through comparative genomics, as demonstrated for L. interrogans strain HP358, which contains several genes known to be virulence factors in other bacterial species, such as Lon protease, cirA (colicin I receptor), and sspH1 (E3 ubiquitin-protein ligase) .

How can researchers overcome solubility issues when expressing recombinant leptospiral proteins?

Protein solubility challenges can be addressed through multiple strategies:

• Expression optimization:

  • Reduce expression temperature (16-25°C)

  • Lower inducer concentration

  • Use specialized E. coli strains (e.g., Rosetta for rare codon usage, SHuffle for disulfide bond formation)

• Fusion partners to enhance solubility:

  • MBP (maltose-binding protein)

  • SUMO

  • Thioredoxin

• Domain-based approaches:

  • Express individual domains separately

  • Remove hydrophobic regions while preserving functional domains

For challenging leptospiral proteins, consider cell-free expression systems that can accommodate toxic or membrane-associated proteins. Alternatively, inclusion body formation followed by refolding can be a viable strategy if native folding can be achieved and verified through functional assays.

What are the best approaches for studying protein-protein interactions among leptospiral proteins in their native environment?

To study protein-protein interactions in native conditions:

• In vivo crosslinking prior to cell lysis to capture transient interactions

• Co-immunoprecipitation using antibodies against native proteins

• Bacterial two-hybrid systems adapted for leptospiral proteins

• Proximity labeling techniques such as BioID or APEX2

For proteins like LIC11051 and LIC11505, cross-reactivity between proteins has been observed and confirmed by ELISA , suggesting potential protein-protein interactions. Such interactions can be further characterized using methods like surface plasmon resonance to determine binding affinities and kinetics, or cryo-electron microscopy for structural characterization of complexes.

How can next-generation sequencing and mass spectrometry approaches advance the study of leptospiral proteins?

Advanced omics technologies offer powerful approaches for leptospiral protein research:

• Next-generation sequencing applications:

  • RNA-Seq to determine expression patterns under different conditions

  • ChIP-Seq to identify regulatory networks controlling protein expression

  • Oxford Nanopore Technologies for direct sequencing of environmental samples

• Mass spectrometry applications:

  • Quantitative proteomics to determine protein abundance (e.g., copies/cell as demonstrated for LIC11051 with an expected 309 copies/cell)

  • Post-translational modification analysis

  • Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification

These methods have led to the identification of novel Leptospira species and improved characterization of protein expression profiles, providing insights into pathogenesis mechanisms and potential diagnostic markers.

What considerations are important when evaluating leptospiral proteins as vaccine candidates?

Evaluation of vaccine potential requires assessment of multiple factors:

• Conservation across pathogenic serovars to ensure broad protection

• Surface exposure or secretion to ensure accessibility to the immune system

• Expression during infection as demonstrated by patient antibody responses

• Minimal sequence similarity to host proteins to avoid autoimmunity

• Ability to induce protective immunity in animal models

For recombinant protein vaccines, immunization studies should evaluate:

• Antibody titers and persistence

• Cellular immune responses

• Protection against challenge with various pathogenic strains

• Safety profile including absence of autoimmune reactions

Leptospiral proteins like rLIC11505, which contains a broad spectrum of ligands including GAG and integrin receptors, and demonstrates expression during infection through antibody recognition in leptospirosis serum samples , might represent promising vaccine candidates pending further evaluation.