Recombinant Chlamydophila caviae GTPase Der (der)

What is GTPase Der in Chlamydophila caviae and what is its biological significance?

GTPase Der (Double-Era-like GTPase) in Chlamydophila caviae is a specialized guanosine triphosphatase that belongs to the family of small GTPases. These proteins function as molecular switches in various cellular processes by cycling between active GTP-bound and inactive GDP-bound states. In bacterial systems like Chlamydia, GTPases play critical roles in ribosome assembly, protein synthesis, and developmental regulation.

The biological significance of Der GTPase stems from its essential function in bacterial survival and replication. While specific research on C. caviae Der is limited, studies on related chlamydial species indicate that bacterial GTPases are often involved in regulating developmental cycles and pathogenesis. For instance, Chlamydiaceae utilize various GTPases during their unique biphasic lifecycle, transitioning between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs) .

What expression systems are most effective for producing recombinant Chlamydophila caviae GTPase Der?

Both E. coli and yeast-based expression systems have been successfully employed for recombinant chlamydial protein production . When working with C. caviae GTPase Der, consider these methodological approaches:

E. coli Expression System:

• Recommended for initial screening due to rapid growth and high protein yields

• Optimal expression typically uses BL21(DE3) strains with pET vector systems

• Induction conditions: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Expression temperature: 18-25°C to enhance proper protein folding

• Addition of 1% glucose to the growth medium helps reduce basal expression

Yeast Expression System:

• Preferred for proteins requiring eukaryotic post-translational modifications

• Pichia pastoris expression produces higher yields for certain chlamydial proteins

• Methanol induction protocol: 0.5% methanol added every 24 hours for 72-96 hours

• Buffered media (pH 6.0) improves stability of the recombinant protein

The choice between these systems should be based on experimental requirements, with E. coli being suitable for structural studies and yeast systems potentially offering better functional activity for enzymatic assays .

How can researchers verify the functional activity of purified recombinant Chlamydophila caviae GTPase Der?

Functional verification requires methodical assessment of GTPase activity. The following protocols are recommended:

Standard GTPase Activity Assay:

• Prepare reaction buffer: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT

• Mix 2-5 μg purified GTPase with 100 μM GTP

• Incubate at 37°C for 15-30 minutes

• Quantify released phosphate using malachite green assay

• Calculate specific activity as nmol Pi released/min/mg protein

Fluorescence-Based Real-time Assay:

• Use BODIPY-FL-GTP as fluorescent substrate

• Monitor decrease in fluorescence (excitation 485 nm, emission 520 nm)

• Reaction conditions: 1 μM enzyme, 0.5 μM BODIPY-FL-GTP

• Record measurements every 30 seconds for 30 minutes

• Determine initial velocity from linear portion of reaction curve

For functional validation, compare activity parameters to those of other bacterial Der GTPases (EC50, Km, kcat). Active recombinant Der should demonstrate GTP hydrolysis rates typically in the range of 5-20 nmol/min/mg protein, with measurable response to physiological regulators.

What purification strategies yield the highest purity and activity for recombinant Chlamydophila caviae GTPase Der?

A multi-step purification approach is essential for obtaining high-purity, functionally active recombinant Der GTPase:

Recommended Purification Protocol:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Binding buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole

  • Wash buffer: Same with 20-40 mM imidazole

  • Elution buffer: Same with 250 mM imidazole

• Intermediate purification: Ion exchange chromatography

  • Buffer A: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • Buffer B: Same with 1 M NaCl

  • Linear gradient: 50-500 mM NaCl over 20 column volumes

• Polishing step: Size exclusion chromatography

  • Running buffer: 20 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • Flow rate: 0.5 ml/min on Superdex 200 column

Throughout purification, maintain 5 mM MgCl₂ in all buffers to stabilize the nucleotide-binding pocket. Addition of 10% glycerol and reducing agents (1-2 mM DTT) helps maintain enzymatic activity. For extended storage, flash-freeze aliquots and store at -80°C with 20% glycerol as cryoprotectant.

How does Chlamydophila caviae GTPase Der differ from other bacterial GTPases?

Chlamydophila caviae GTPase Der possesses distinct characteristics that differentiate it from other bacterial GTPases:

Unlike other GTPases involved in host-pathogen interactions (like Rab GTPases that interact with chlamydial inclusion membrane proteins), Der GTPase predominantly functions in bacterial physiology . The protein's distinct structure with two GTP-binding domains (G domains) arranged in tandem allows for unique regulatory mechanisms not found in single-domain GTPases like those in the Ras superfamily described in the literature .

What experimental approaches can be used to study the interaction between Chlamydophila caviae GTPase Der and potential binding partners?

Several methodological approaches can be employed to investigate Der GTPase interactions:

Yeast Two-Hybrid System:

• Clone Der coding sequence into bait vector (DNA-binding domain fusion)

• Screen against chlamydial genomic library in prey vector (activation domain fusion)

• Select for positive interactions using appropriate auxotrophic markers

• Confirm with direct mating assays using individual clones

• Validate interactions using secondary screens (co-IP, pull-down)

This approach successfully identified interactions between chlamydial inclusion membrane protein Cpn0585 and various Rab GTPases, and could be adapted for Der GTPase studies .

GST Pull-down Assays:

• Express GST-tagged Der GTPase in E. coli

• Immobilize on glutathione-sepharose beads

• Prepare bacterial or host cell lysates

• Incubate immobilized protein with lysates

• Wash extensively and elute bound proteins

• Analyze by SDS-PAGE and mass spectrometry

For studying nucleotide dependence of interactions (common in GTPase biology), perform parallel experiments with Der locked in GTP-bound (active) or GDP-bound (inactive) states using point mutations at the catalytic site .

Fluorescence Co-localization:

• Generate fluorescently tagged Der constructs (e.g., EGFP fusion)

• Transfect into appropriate cell models

• Infect with C. caviae

• Perform immunofluorescence for potential partner proteins

• Analyze co-localization using confocal microscopy

This approach has been successful in studying the localization of EGFP-tagged Rab GTPases in relation to chlamydial inclusion proteins .

How can researchers develop inhibitors targeting Chlamydophila caviae GTPase Der?

A systematic approach to developing Der GTPase inhibitors involves:

Structure-Based Inhibitor Design:

• Determine crystal structure of C. caviae Der using X-ray crystallography

  • Expression of recombinant protein with hexa-histidine tag

  • Purification by affinity chromatography followed by size exclusion

  • Crystallization screening in presence of non-hydrolyzable GTP analogs

• Identify nucleotide-binding pocket and unique structural features

• Perform in silico screening of compound libraries against identified binding sites

• Validate top hits with in vitro GTPase activity assays

• Optimize lead compounds through medicinal chemistry approaches

High-Throughput Screening:

• Develop fluorescence-based or colorimetric GTPase activity assay adapted to 384-well format

• Screen compound libraries (10,000-100,000 compounds)

• Confirm hits with dose-response curves

• Evaluate specificity against human GTPases

• Assess antibacterial activity in infected cell models

GTP-Competitive Inhibitor Design:

• Synthesize GTP analogs with modifications at ribose, phosphate, or base moieties

• Test binding affinity using fluorescence polarization assays

• Evaluate inhibition of GTPase activity using malachite green phosphate detection

• Determine mechanism of inhibition through enzyme kinetics studies

• Assess cellular permeability and antimicrobial activity

This approach parallels successful development of inhibitors targeting bacterial GTPases in other pathogens while focusing on the unique features of Der GTPase.

How do mutations in GTPase Der affect Chlamydophila caviae survival and development?

Understanding the impact of Der mutations requires specialized genetic approaches:

Methodological Approach to Der Mutational Analysis:

• Generate conditional mutants using TargeTron or FRAEM technologies

  • These methods have been successfully applied to create chlamydial mutants

  • Design insertion sites in conserved GTPase motifs (G1-G5)

  • Create both null mutations and point mutations affecting GTP binding/hydrolysis

• Introduce mutations into C. caviae genome

• Assess impact on bacterial growth using inclusion size measurement

• Evaluate developmental cycle progression using stage-specific markers

• Perform electron microscopy to identify morphological abnormalities

• Conduct competition assays with wild-type bacteria

Expected Phenotypes Based on Der Function:

• Mutations in GTP-binding motifs likely produce severe growth defects

• Hydrolysis-deficient mutants may exhibit altered developmental timing

• Domain-specific mutations can reveal differential functions of N- and C-terminal G domains

• Temperature-sensitive mutations could allow temporal control of Der function

Recent advances in chlamydial genetics, including the development of techniques like TargeTron, have facilitated the creation of directed mutants, allowing researchers to study essential genes like Der through conditional approaches .

How can researchers effectively study the impact of Der GTPase inhibition on chlamydial infection in different cell types?

A comprehensive approach to studying Der GTPase inhibition requires:

Experimental Design for Der Inhibition Studies:

• Develop cellular models representing different infection sites

  • HeLa cells (standard laboratory model)

  • Primary epithelial cells (more physiologically relevant)

  • Polarized cell systems (to model epithelial barriers)

• Establish Der inhibition methods

  • Small molecule inhibitors identified through screening

  • Conditional genetic systems (if available)

  • RNA interference approaches targeting Der expression

• Implement infection protocols

  • Standardize infection MOI (0.5-1 for single-cell analysis)

  • Establish time course (0-48 hours post-infection)

  • Control for cell type-specific responses

Analytical Methods:

• Quantitative assessment of inclusion development

  • Automated microscopy with image analysis

  • Flow cytometry of infected cells

  • Inclusion size measurement

• Bacterial replication quantification

  • qPCR targeting chlamydial genes

  • Infectious progeny recovery assays

  • Genomic copy number determination

• Host-pathogen interaction analysis

  • Transcriptomics of host response

  • Phosphoproteomic analysis of signaling pathways

  • Cytokine/chemokine profiling

This approach allows for systematic comparison of Der inhibition effects across cell types, revealing potential tissue-specific responses to bacterial GTPase targeting .

What is the role of GTPase Der in Chlamydophila caviae stress response and antibiotic resistance?

The involvement of Der GTPase in stress response mechanisms and potential antibiotic resistance remains an important research question:

Methodological Approach to Stress Response Studies:

• Expose C. caviae cultures to various stressors

  • Nutrient limitation (amino acid starvation)

  • Oxidative stress (H₂O₂ treatment)

  • Temperature shifts (heat shock)

  • Antibiotic pressure (sub-inhibitory concentrations)

• Quantify Der expression levels

  • qRT-PCR for transcriptional changes

  • Western blot for protein levels

  • Reporter gene fusions to monitor promoter activity

• Analyze Der activation state

  • GTP/GDP binding ratio determination

  • Co-immunoprecipitation with known binding partners

  • Phosphorylation status assessment

Antibiotic Resistance Studies:

• Generate C. caviae strains with modulated Der expression

• Determine minimum inhibitory concentrations (MICs) for various antibiotics

• Assess development of resistance under selective pressure

• Evaluate ribosome profiles in Der-modulated strains

• Analyze translation fidelity and efficiency