Recombinant Chlamydophila caviae 30S ribosomal protein S7 (rpsG)

What is the structure and biochemical composition of Chlamydophila caviae 30S ribosomal protein S7?

The Chlamydophila caviae 30S ribosomal protein S7 (rpsG) is a full-length protein consisting of 157 amino acids. According to available recombinant protein specifications, the complete amino acid sequence is:

MSRRHAAEKK VIPADPIYGS VTLERFINKV MMHGKKSIAR KIVYSALERF SKKIGAENVL EAFKEALENA KPLLEVRSRR VGGATYQVPV EVAAGRRDCL AMKWIINNAR NKPGKCMEVG LATELIDCFN KQGATIKKRE DTHRMAEANK AFAHYKW

The protein is encoded by the rpsG gene (Gene ID: CCA_0019130) in Chlamydophila caviae strain GPIC. As a component of the small (30S) ribosomal subunit, S7 likely adopts a three-dimensional structure that enables its functional interactions with ribosomal RNA and other ribosomal proteins within the assembled ribosome .

What functional roles does the S7 protein play in the ribosomal complex?

Based on research with bacterial ribosomal proteins, S7 serves several critical functions in the ribosomal complex:

• It forms an essential connection between the head of the 30S subunit and the platform region via interaction with ribosomal protein S11 .

• This interaction is located in the E site of the ribosome and participates in forming the exit channel through which mRNA passes during translation .

• S7 contributes to the control of translational fidelity, as mutations in this protein can lead to increased frameshifting, readthrough of nonsense codons, and codon misreading .

• The protein plays a crucial role in 30S subunit assembly, serving as a nucleation center for ribosome biogenesis .

These functions highlight S7's importance in both the structural integrity and functional activity of the ribosome.

What expression systems are available for recombinant production of C. caviae S7 protein?

Several expression systems can be utilized for the production of recombinant C. caviae 30S ribosomal protein S7, each with distinct advantages depending on research requirements:

The choice of expression system should be guided by the specific experimental requirements, including the need for post-translational modifications, protein folding considerations, and downstream applications .

What purification methods yield high-quality recombinant S7 protein for functional studies?

While specific purification protocols for C. caviae S7 aren't detailed in the search results, recombinant S7 proteins are typically purified to >85% purity as determined by SDS-PAGE . Based on methodologies used for other ribosomal proteins, a recommended purification approach would include:

• Initial capture using affinity chromatography (dependent on the fusion tag employed)

• Tag removal if applicable (using proteases specific to engineered cleavage sites)

• Secondary purification via ion exchange chromatography

• Polishing step using size exclusion chromatography

• Quality assessment via SDS-PAGE and potentially mass spectrometry

For functional studies, the final product is typically provided as a lyophilized powder, which requires proper reconstitution in an appropriate buffer before use . Researchers should centrifuge the vial briefly before opening to ensure all material is collected at the bottom of the container.

How can researchers reconstitute functional 30S ribosomal subunits using recombinant S7 protein?

In vitro reconstitution of 30S ribosomal subunits represents an advanced application of recombinant ribosomal proteins like S7. Based on established protocols, the following methodology can be adapted for C. caviae S7:

• Component preparation: Purify individual ribosomal proteins (S2-S21) using methods such as SUMO fusion for optimal solubility and native structure .

• Assembly under high-salt conditions:

  • Combine purified 16S rRNA with recombinant ribosomal proteins including S7

  • Incubate under high-salt conditions (typically 330 mM KCl, 20 mM MgCl₂)

  • Perform heat activation at 42°C to facilitate proper assembly

• Alternative physiological reconstitution:

  • Assembly can also be performed under more physiological salt conditions (150 mM K⁺, 5 mM Mg²⁺) when supplemented with ribosome biogenesis factors

  • Biogenesis factors such as Era, YjeQ, and RimP enhance assembly efficiency, with Era showing the strongest effect

• Functional assessment:

  • Evaluate reconstituted subunits using poly(U)-directed polyphenylalanine synthesis assays

  • For complete functional assessment, test in the PURE system for full-length protein synthesis

  • The addition of S1 protein significantly enhances activity (approximately twofold increase)

Reconstituted 30S subunits containing all necessary proteins typically exhibit approximately 30% of the activity of native 30S subunits, which can increase to ~80% with the addition of S1 protein .

What experimental approaches can reveal the functional interaction between S7 and other ribosomal proteins?

Based on bacterial ribosome studies, S7 forms critical interactions with other ribosomal proteins, particularly S11. These interactions can be studied through several experimental approaches:

• Site-directed mutagenesis:

  • Design mutations targeting residues at the S7-S11 interface based on structural data

  • Introduce these mutations into recombinant S7 protein

  • Assess the impact on ribosome assembly and function

• Functional assays to assess translational fidelity:

  • In vivo assays measuring frameshifting efficiency

  • Nonsense codon readthrough assays

  • Misreading assays to quantify amino acid misincorporation rates

• mRNA binding assessment:

  • Toeprinting assays to measure the position of ribosomes on mRNA

  • Filter-binding assays to quantify mRNA binding capacity

Research has shown that disrupting the S7-S11 interaction results in altered translational fidelity, with increased capacity for frameshifting, readthrough of nonsense codons, and codon misreading. Additionally, 30S subunits with mutated S7 or S11 demonstrate enhanced capacity to bind mRNA . These effects can be attributed to:

• Increased flexibility of the 30S subunit head

• Opening of the mRNA exit channel

• Perturbation of the allosteric coupling between A and E sites

How does the sequence and structure of C. caviae S7 compare to S7 proteins from other bacterial species?

Comparative analysis of S7 proteins across bacterial species can provide insights into evolutionary conservation and species-specific adaptations. While comprehensive comparative data is not provided in the search results, researchers can approach this question through:

• Sequence alignment analysis:

  • Align the C. caviae S7 sequence (157 amino acids) with S7 sequences from model organisms like E. coli

  • Identify conserved regions that may be critical for function

  • Note divergent regions that might reflect species-specific adaptations

• Structural prediction and comparison:

  • Generate structural models of C. caviae S7 using homology modeling

  • Compare predicted structures with existing crystal structures of S7 from other bacteria

  • Analyze the conservation of key interaction surfaces, particularly those involved in binding to S11, rRNA, or mRNA

• Functional domain mapping:

  • Identify domains responsible for ribosome incorporation

  • Map regions involved in translational fidelity control

  • Determine species-specific structural elements that might contribute to pathogen-specific ribosomal function

This comparative approach can reveal evolutionary pressures on ribosomal proteins in different bacterial lineages and potentially identify unique features of C. caviae S7 that might be related to its intracellular lifestyle as an obligate pathogen .

What genetic tools are available for studying S7 function in Chlamydophila species?

Studying ribosomal proteins like S7 in Chlamydophila species has historically been challenging due to the limited genetic tools available for these obligate intracellular pathogens. Recent developments have improved this situation:

• Shuttle vector-based transformation systems:

  • Recently developed for C. caviae, enabling genetic manipulation

  • Allow for the potential expression of tagged versions of ribosomal proteins

  • Enable creation of conditional mutants to study essential proteins like S7

• Heterologous expression and complementation:

  • Express C. caviae S7 in model organisms like E. coli

  • Perform complementation studies to assess functional conservation

  • Utilize ribosome assembly assays with heterologous components

• Reverse genetics approaches:

  • CRISPR-based technologies adapted for Chlamydia

  • Targeted mutagenesis to study specific domains of S7

  • Conditional expression systems to study essential genes

These genetic tools open new avenues for investigating the role of S7 in Chlamydophila biology, including its potential contributions to pathogenesis and adaptation to the intracellular lifestyle .

What is the potential significance of S7 protein in Chlamydophila pathogenesis?

While the search results don't directly address the role of S7 in pathogenesis, several potential research directions can be considered:

Understanding the role of S7 in Chlamydophila caviae, known to cause conjunctivitis in guinea pigs and pneumonia in humans, could provide insights into the basic biology of this pathogen and potentially reveal new therapeutic approaches .

What quality control measures should be implemented when working with recombinant S7 protein?

Ensuring the quality and functionality of recombinant C. caviae S7 protein is critical for experimental success. Recommended quality control measures include:

• Purity assessment:

  • SDS-PAGE analysis (expect >85% purity)

  • Mass spectrometry to confirm protein identity

  • Endotoxin testing if intended for cell-based assays

• Structural integrity evaluation:

  • Circular dichroism to assess secondary structure

  • Limited proteolysis to verify proper folding

  • Size exclusion chromatography to confirm monomeric state

• Functional verification:

  • RNA binding assays

  • Incorporation into reconstituted 30S subunits

  • Assessment of reconstituted ribosome activity using translation assays

• Storage and handling precautions:

  • Store lyophilized protein at -20°C

  • Reconstitute carefully following manufacturer recommendations

  • Avoid repeated freeze-thaw cycles of reconstituted protein

Implementation of these quality control measures helps ensure experimental reproducibility and validity when working with this complex ribosomal protein.

How can researchers optimize reconstitution conditions for maximum S7 functionality?

Optimal reconstitution of lyophilized S7 protein is crucial for maintaining its functional properties. Based on general protein reconstitution principles and the specific nature of ribosomal proteins:

• Buffer selection:

  • Use buffers mimicking physiological conditions (pH 7.4-7.6)

  • Include stabilizing components such as glycerol (10-30%)

  • Consider adding reducing agents like DTT (1mM) to maintain cysteine residues in reduced state

• Reconstitution procedure:

  • Centrifuge the vial briefly before opening to collect all material

  • Add buffer slowly while gently rotating the vial

  • Avoid vigorous vortexing which can cause protein denaturation

  • Allow complete dissolution before use

• Concentration optimization:

  • For ribosome reconstitution studies, protein concentration typically ranges from 0.5-2 mg/ml

  • Higher concentrations may be needed for structural studies

  • Lower concentrations may be preferable for interaction studies to minimize aggregation

• Validation of reconstituted protein:

  • Verify solubility by centrifugation to remove any insoluble material

  • Confirm activity using appropriate functional assays before proceeding with main experiments

Following these optimization strategies can significantly improve the success rate of experiments utilizing recombinant S7 protein.