Recombinant Bordetella bronchiseptica 50S ribosomal protein L10 (rplJ)
Description
Introduction
The recombinant Bordetella bronchiseptica 50S ribosomal protein L10 (rplJ) is a component of the large ribosomal subunit, specifically the 50S subunit, in the bacterium Bordetella bronchiseptica . Ribosomal proteins like rplJ are crucial for protein synthesis, as they contribute to the structure and function of the ribosome . More specifically, RplJ forms part of the ribosomal stalk, which helps the ribosome interact with GTP-bound translation factors .
Characteristics of rplJ
RplJ, also known as the large ribosomal subunit protein uL10, plays a central role in the interaction of the ribosome with GTP-bound translation factors . It belongs to the universal ribosomal protein uL10 family . The Mycoplasma mycoides rplJ protein is 165 amino acids long . The Bradyrhizobium sp. ORS278 rplJ protein is 172 amino acids long .
Function in Protein Synthesis
RplJ is essential for accurate translation because it forms part of the ribosomal stalk . It plays a role in the coordinated movement of tRNA molecules and mRNA, as well as conformational changes within the ribosome during protein synthesis .
rplJ as a Vaccine Target
Researchers have explored the potential of B. bronchiseptica proteins, including recombinant forms, as vaccine candidates . Studies have focused on identifying proteins that can induce immune responses in animals, with the goal of developing vaccines to prevent respiratory infections caused by this bacterium .
Immune Response to Recombinant Proteins
Recombinant proteins from B. bronchiseptica can induce high antibody titers in mice . Some proteins, such as outer membrane porin protein (PPP) and lipoprotein (PL), have demonstrated immune-protective potential against B. bronchiseptica challenges . These proteins can induce both humoral and cell-mediated immune responses, with a dominant Th2-type response .
Table 1. Splenocyte proliferation and cytokine analysis of immune-protective proteins
| Group (n = 3) | Cytokine concentration (pg/ml) a | SI b |
|---|---|---|
| IFN-γ | IL-2 | |
| rPPP | 625.92 ± 5.91* | 18.54 ± 1.41* |
| rPL | 676.07 ± 5.52* | 17.91 ± 0.28* |
| Negative control | 546.97 ± 9.39 | 14.76 ± 0.97 |
Splenocytes were harvested from the mice 2 weeks after the second immunization with rPPP or rPL. Data represent the mean ± SD from three independent experiments; n = 3 mice per group. Single-cell suspensions of splenocytes were stimulated with an optimized concentration of corresponding recombinant protein. P < 0.05 compared to the control groups
a Values for IL-2, IL-4, IL-10, and IFN-γ are titers at 60 h
b SI stands for stimulation index
Interaction with Other Ribosomal Proteins
RplJ interacts with several other ribosomal proteins, which contributes to the assembly and function of the ribosome . These proteins include:
Product Specs
Target Background
This protein is a component of the ribosomal stalk, playing a crucial role in the ribosome's interaction with GTP-bound translation factors.
KEGG: bbr:BB0012
STRING: 257310.BB0012
Q&A
What is the biological function of 50S ribosomal protein L10 (rplJ) in Bordetella bronchiseptica?
The 50S ribosomal protein L10 (rplJ) from Bordetella bronchiseptica forms part of the ribosomal stalk, playing a central role in the interaction of the ribosome with GTP-bound translation factors . It belongs to the universal ribosomal protein uL10 family, which is highly conserved across bacterial species . Similar to its homologs in other bacteria, rplJ in B. bronchiseptica is essential for ribosome assembly and protein synthesis, serving as a crucial component of the translation machinery. Functional studies have demonstrated that rplJ acts as an interface between the ribosome and translation factors, facilitating the GTPase activity required for protein elongation.
How does rplJ compare to homologous proteins in other bacterial species?
rplJ proteins are highly conserved across bacterial species, reflecting their essential role in translation. Comparative analysis reveals significant sequence similarity between B. bronchiseptica rplJ and homologs from other species:
| Species | Length (aa) | Sequence Identity | Mass (kDa) |
|---|---|---|---|
| B. bronchiseptica RB50 | 174 | 100% (reference) | 18.5 |
| Shigella flexneri | 165 | ~60% | 17.7 |
| E. coli | 165 | ~60% | 17.7 |
| Other Bordetella spp. | 172-175 | 90-98% | 18.3-18.7 |
Despite variations in sequence, the functional domains remain conserved, particularly those involved in ribosome binding and interaction with translation factors. Phylogenetic analyses suggest that rplJ evolution closely mirrors the evolutionary relationships between bacterial species, making it a useful marker for taxonomic studies .
What are the optimal expression systems for producing recombinant B. bronchiseptica rplJ protein?
Multiple expression systems can be utilized for producing recombinant B. bronchiseptica rplJ, each with distinct advantages depending on research needs:
| Expression System | Advantages | Yield | Purification Strategy | Applications |
|---|---|---|---|---|
| E. coli | Cost-effective, rapid growth, high yield | 5-10 mg/L | IMAC via His-tag, Size exclusion | Structural studies, antibody production |
| Yeast | Post-translational modifications, proper folding | 3-7 mg/L | Affinity chromatography | Functional assays |
| Baculovirus | Complex folding, higher eukaryotic PTMs | 2-5 mg/L | Affinity tags, ion exchange | Interaction studies |
| Mammalian | Native-like folding and modifications | 1-3 mg/L | Immunoaffinity, FPLC | In vivo functional studies |
For most structural and biochemical characterizations, E. coli-based expression (particularly BL21(DE3) strain) with an N-terminal His-tag provides optimal yields and purity . For expression in E. coli, using the pET vector system with IPTG induction at 18°C overnight after reaching OD600 of 0.6-0.8 typically yields soluble protein. Protein can be efficiently purified using immobilized metal affinity chromatography followed by size exclusion chromatography to remove aggregates and obtain >95% purity required for structural or functional studies .
What methodologies are most effective for studying rplJ protein-protein interactions within the ribosome?
Several complementary approaches can be employed to study rplJ interactions:
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Co-immunoprecipitation (Co-IP): Using antibodies against rplJ to pull down associated proteins followed by mass spectrometry analysis. This approach has identified interactions between rplJ and other ribosomal proteins including L7/L12 stalk proteins.
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Yeast two-hybrid assays: Though challenged by the complexity of ribosomal assemblies, modified Y2H approaches using domain-specific constructs have successfully identified binary interactions.
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Cryo-electron microscopy: For visualizing rplJ within the whole ribosomal complex, providing context for its interactions and conformational changes during translation .
These methodologies have revealed that rplJ interacts primarily with the 23S rRNA and forms part of the GTPase-associated center, serving as an anchor point for the ribosomal stalk proteins that recruit translation factors to the ribosome.
What approaches can be used to study the role of rplJ in B. bronchiseptica iron starvation response pathways?
Research has shown that iron limitation affects ribosomal protein expression in Bordetella species, including potential regulatory roles for rplJ. A comprehensive approach to investigate this relationship includes:
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Transcriptional profiling: RNA isolation from B. bronchiseptica grown under iron-replete and iron-depleted conditions followed by microarray or RNA-seq analysis, as demonstrated in previous studies . This reveals the transcriptional regulation of rplJ under iron limitation.
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Protein expression analysis: Comparative proteomics using 2D-PAGE or LC-MS/MS to quantify rplJ protein levels under different iron conditions .
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Chromatin immunoprecipitation (ChIP): To identify potential regulatory proteins that bind to the rplJ promoter region under iron limitation.
Research has demonstrated that iron starvation in B. bronchiseptica alters the expression of numerous proteins, including components of the Type III Secretion System (T3SS) . Investigation of potential functional interactions between rplJ and the iron-responsive regulatory networks could reveal novel insights into stress adaptation mechanisms in this pathogen.
How can researchers design experiments to elucidate the potential role of rplJ in antibiotic resistance mechanisms?
Given that ribosomal proteins are targets for several antibiotics, investigating rplJ's role in resistance requires multi-faceted approaches:
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Structural biology approaches: X-ray crystallography or cryo-EM to visualize antibiotic binding sites on the ribosome, particularly interactions with rplJ. These studies have revealed that macrolides like roxithromycin and clarithromycin, which target the 50S ribosomal subunit, can interact with regions near rplJ .
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Antibiotic selection experiments: Passaging B. bronchiseptica in sub-inhibitory concentrations of antibiotics targeting the 50S ribosomal subunit (macrolides, lincosamides) followed by whole genome sequencing to identify potential mutations in rplJ associated with resistance.
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Heterologous expression assays: Expressing wild-type or mutant rplJ variants in antibiotic-sensitive E. coli strains to determine if specific rplJ variants confer resistance.
These approaches can help elucidate whether rplJ mutations contribute to the significant antibiotic resistance observed in some B. bronchiseptica strains, particularly to macrolides which target the 50S ribosomal subunit.
What methodological approaches are optimal for investigating post-translational modifications of rplJ and their functional significance?
Post-translational modifications (PTMs) of ribosomal proteins, including rplJ, can significantly impact ribosome assembly, translation efficiency, and antibiotic sensitivity. Research methodologies to investigate PTMs include:
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Site-specific antibodies: Generating antibodies against predicted modified epitopes of rplJ to detect specific PTMs in different growth conditions.
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In vitro modification assays: Identifying enzymes responsible for rplJ modifications using purified proteins and relevant cofactors, followed by functional characterization.
Common PTMs found on bacterial ribosomal proteins include methylation, acetylation, and phosphorylation. For rplJ specifically, methylation at conserved lysine residues has been reported in other bacterial species, potentially affecting ribosome assembly and translation efficiency under stress conditions .
What research design would best investigate the potential of rplJ as a vaccine target against B. bronchiseptica infections?
Evaluating rplJ as a vaccine candidate requires a systematic approach:
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Immunogenicity assessment:
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Express and purify recombinant rplJ with proper folding
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Analyze B-cell epitope prediction using algorithms like BepiPred
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Perform T-cell epitope mapping using overlapping peptides
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Evaluate cross-reactivity with host proteins through sequence alignment and immunological assays
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Accessibility studies: Using immunofluorescence microscopy and flow cytometry to determine if antibodies against rplJ can access their target in intact bacteria.
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Functional neutralization assays: Evaluating whether anti-rplJ antibodies can inhibit bacterial growth in vitro through complement-dependent mechanisms or by interfering with essential functions.
