Recombinant Chromobacterium violaceum 50S ribosomal protein L25 (rplY)

How does the amino acid sequence of C. violaceum rplY compare to homologous proteins in other bacterial species?

C. violaceum rplY shares significant sequence homology with L25 proteins from other beta-proteobacteria. Sequence alignment analyses reveal conserved domains that are involved in RNA binding and ribosome assembly. The protein contains specific motifs characteristic of the L25 family, particularly in regions that interact with 5S rRNA . While the core functional domains show high conservation across bacterial species, C. violaceum rplY exhibits some unique sequence variations that may reflect adaptations to the tropical environments where this bacterium naturally thrives.

What expression systems are most suitable for producing recombinant C. violaceum rplY?

E. coli-based expression systems are most commonly used for the recombinant production of C. violaceum rplY due to their high efficiency and ease of manipulation. Based on experimental findings with other recombinant proteins from C. violaceum, BL21(DE3) or Rosetta strains are particularly effective when the gene is cloned into vectors containing T7 promoters . The expression conditions should be optimized, with induction at lower temperatures (25°C instead of 37°C) and moderate IPTG concentrations (0.1-0.5 mM) to enhance soluble protein production . Alternative systems such as Bacillus subtilis may be considered for certain applications, particularly if post-translational modifications are required.

What factorial design approach should be used to optimize recombinant C. violaceum rplY expression?

A 2^8-4 fractional factorial design is recommended to efficiently optimize rplY expression while minimizing the number of experiments required. This approach allows for systematic evaluation of multiple variables affecting protein expression simultaneously . Eight key variables should be tested:

• Induction temperature (25°C vs. 37°C)

• IPTG concentration (0.1 mM vs. 1.0 mM)

• Post-induction time (4h vs. 18h)

• Cell density at induction (OD600 of 0.6 vs. 1.2)

• Media composition (minimal vs. rich)

• pH (6.8 vs. 7.5)

• Glucose concentration (0 vs. 1 g/L)

• Oxygen transfer rate (low vs. high)

Results from similar experimental design studies with other recombinant proteins from C. violaceum indicate that an optimized protocol typically involves induction at OD600 of 0.8, using 0.1 mM IPTG, with expression at 25°C for 4 hours in a medium containing 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, and 1 g/L glucose .

How can I assess the solubility of recombinant C. violaceum rplY during optimization?

The solubility of recombinant rplY should be assessed through a systematic fractionation approach combined with quantitative analysis. After cell lysis, separate the total cell extract, soluble fraction, and insoluble fraction by centrifugation. Analyze each fraction using SDS-PAGE followed by densitometry to quantify the distribution of rplY in each fraction . For accurate assessment, use the following protocol:

Calculate the percentage of soluble rplY by dividing the band intensity in the soluble fraction by the sum of intensities in both soluble and insoluble fractions. Optimized conditions typically yield >60% of the protein in the soluble fraction .

What strategies can mitigate inclusion body formation when expressing rplY?

Several strategies can be implemented to reduce inclusion body formation and enhance soluble expression of rplY:

• Temperature reduction: Lower the post-induction temperature to 25°C or even 16°C to slow protein synthesis and allow proper folding .

• Co-expression with chaperones: Co-transform the expression host with plasmids encoding chaperones such as GroEL/GroES or DnaK/DnaJ/GrpE.

• Fusion partners: Employ solubility-enhancing fusion tags such as MBP (maltose-binding protein), SUMO, or Thioredoxin.

• Medium optimization: Add osmolytes like sorbitol (0.5 M) and glycylglycine (1 mM) to the culture medium to stabilize protein structure.

• Pulse induction: Use lower IPTG concentrations (0.1 mM) with extended expression times rather than high inducer concentrations .

Research has shown that combining temperature reduction (25°C) with moderate IPTG concentration (0.1 mM) can increase soluble rplY yield by up to 3-fold compared to standard conditions .

What is the most efficient purification strategy for recombinant C. violaceum rplY?

A multi-step purification approach is recommended for obtaining high-purity recombinant C. violaceum rplY:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin if the protein is expressed with a His-tag. Optimize elution with an imidazole gradient (20-250 mM).

• Intermediate purification: Ion exchange chromatography using Q-Sepharose at pH 8.0 (above the theoretical pI of rplY).

• Polishing step: Size exclusion chromatography using Superdex 75 or 200 columns to remove aggregates and achieve >95% purity.

This strategy typically yields approximately 40-50 mg of purified rplY per liter of culture with >95% homogeneity, as determined by SDS-PAGE and densitometry analysis . For structural studies requiring exceptionally pure protein, an additional reverse-phase HPLC step may be necessary.

How can I verify the folding integrity of purified recombinant C. violaceum rplY?

Proper folding of rplY can be assessed using multiple complementary techniques:

• Circular Dichroism (CD) spectroscopy: Analyze secondary structure content by obtaining CD spectra in the far-UV range (190-260 nm). Properly folded rplY should display characteristic minima at 208 and 222 nm reflecting its alpha-helical content.

• Fluorescence spectroscopy: Measure intrinsic tryptophan fluorescence (excitation at 280 nm, emission scan 300-400 nm) to assess tertiary structure.

• Thermal shift assay: Determine protein stability by monitoring unfolding using SYPRO Orange dye in a real-time PCR instrument.

• Functional assay: Verify RNA binding capacity using electrophoretic mobility shift assays (EMSA) with 5S rRNA.

The integration of these methods provides comprehensive validation of proper protein folding, which is essential before proceeding to structural or interaction studies.

What analytical methods are most suitable for characterizing recombinant C. violaceum rplY?

Multiple analytical methods should be employed for comprehensive characterization:

These methods provide critical information about protein quality and are essential for ensuring reproducible results in downstream applications such as structural studies or protein-RNA interaction analyses .

What crystallization conditions are optimal for structural studies of C. violaceum rplY?

Based on successful crystallization of similar ribosomal proteins, the following conditions are recommended as starting points for rplY crystallization trials:

• Vapor diffusion method: Sitting or hanging drop with 1:1 ratio of protein:reservoir solution

• Protein concentration: 8-12 mg/mL in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

• Initial screening: Commercial sparse matrix screens (Hampton Crystal Screen, Molecular Dimensions PACT premier)

• Optimization conditions:

  • PEG-based conditions: 15-25% PEG 3350, 0.1 M MES pH 6.0-6.5, 0.2 M ammonium sulfate

  • Salt-based conditions: 1.8-2.2 M ammonium sulfate, 0.1 M Bis-Tris pH 5.5-6.5

• Additives: 10 mM MgCl2 or 5% glycerol to improve crystal quality

Crystals typically appear within 3-7 days at 18°C and should diffract to at least 2.5 Å resolution for meaningful structural analysis. Co-crystallization with 5S rRNA fragments may stabilize the protein and yield better-diffracting crystals.

How can I design experiments to study the RNA binding properties of C. violaceum rplY?

RNA binding studies for C. violaceum rplY should include multiple complementary techniques:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate increasing concentrations of purified rplY (0.1-10 μM) with labeled 5S rRNA

  • Analyze complex formation on 6% non-denaturing polyacrylamide gels

  • Calculate binding affinities from densitometric analysis

• Isothermal Titration Calorimetry (ITC):

  • Titrate rplY (200-300 μM) into 5S rRNA solution (20-30 μM)

  • Determine thermodynamic parameters (ΔH, ΔS, Kd)

  • Typical binding affinity (Kd) for ribosomal protein-RNA interactions ranges from 10^-7 to 10^-9 M

• Fluorescence anisotropy:

  • Label 5S rRNA with fluorescent dye (e.g., fluorescein)

  • Measure anisotropy changes upon titration with rplY

  • Derive binding curves and calculate dissociation constants

• Surface Plasmon Resonance (SPR):

  • Immobilize biotinylated 5S rRNA on streptavidin sensor chip

  • Flow rplY at different concentrations (1-500 nM)

  • Analyze association and dissociation kinetics (kon, koff)

These methodologies provide comprehensive characterization of the binding interaction, including affinity, stoichiometry, and binding kinetics .

What computational approaches can predict functional sites in C. violaceum rplY?

Several computational approaches can effectively predict functional sites in rplY:

• Sequence conservation analysis:

  • Multiple sequence alignment of L25 family proteins

  • Identification of highly conserved residues using ConSurf or SIFT

  • Typical results show conservation clusters in RNA binding regions

• Structure-based prediction:

  • Homology modeling based on existing L25 structures

  • Electrostatic surface potential calculation to identify positively charged patches (potential RNA binding sites)

  • Normal mode analysis to identify flexible regions involved in binding dynamics

• Machine learning approaches:

  • Random forest algorithms for binding site prediction

  • Support vector machines for protein-RNA interaction site prediction

  • Deep learning models integrating sequence and structural features

• Molecular dynamics simulations:

  • 100-200 ns simulations to identify structurally stable regions

  • Principal component analysis to characterize conformational states

  • Identify water-mediated hydrogen bond networks essential for RNA recognition

These computational predictions should be validated experimentally through site-directed mutagenesis followed by functional assays to confirm the role of predicted residues in RNA binding or structural stability .

How can C. violaceum rplY be used for studying ribosome assembly mechanisms?

C. violaceum rplY provides an excellent model for investigating ribosome assembly mechanisms through several experimental approaches:

• In vitro reconstitution assays:

  • Purify individual ribosomal components (rRNAs and r-proteins)

  • Systematically assemble sub-complexes with and without rplY

  • Monitor assembly using sucrose gradient centrifugation

  • Quantify impact of rplY on assembly efficiency and kinetics

• Time-resolved structural studies:

  • Use cryo-electron microscopy to capture assembly intermediates

  • Incorporate rplY at different time points during assembly

  • Track structural changes associated with rplY incorporation

• Fluorescence-based approaches:

  • Label rplY with donor fluorophore and other r-proteins with acceptor

  • Monitor FRET signals during assembly to map interaction networks

  • Determine order and kinetics of assembly steps

• Genetic complementation:

  • Create conditional knockdown of native rplY in C. violaceum

  • Complement with mutant variants to identify essential functional domains

  • Analyze polysome profiles to assess impact on ribosome biogenesis

These approaches provide insights into the role of rplY in ribosome assembly pathways and help elucidate general principles of macromolecular complex formation .

What techniques can assess differences between native and recombinant C. violaceum rplY?

Comprehensive comparison between native and recombinant rplY requires multiple analytical techniques:

Research findings typically show that carefully expressed and purified recombinant rplY retains most structural and functional properties of the native protein, with differences primarily in post-translational modifications that may affect protein stability but not core functionality .

How can C. violaceum rplY be used in cross-linking studies to map ribosomal protein-RNA interactions?

Cross-linking studies with C. violaceum rplY can map precise protein-RNA contact points using the following methodologies:

• UV cross-linking:

  • Irradiate rplY-RNA complexes with UV light (254 nm)

  • Digest with RNases to leave only cross-linked nucleotides

  • Identify cross-linked residues by mass spectrometry

  • Typical results identify aromatic amino acids (Phe, Tyr, Trp) at RNA binding interfaces

• Chemical cross-linking:

  • Use heterobifunctional cross-linkers (e.g., EDC) to form covalent bonds

  • Optimize cross-linker concentration (0.1-5 mM) and reaction time

  • Analyze by LC-MS/MS to identify cross-linked peptide-RNA adducts

  • Map interaction sites to 3D structural models

• Photo-activatable nucleotide analogs:

  • Incorporate 4-thiouridine or 6-thioguanosine into RNA

  • Activate cross-linking with long-wavelength UV (365 nm)

  • Identify zero-length cross-links representing direct contacts

• Hydrogen-deuterium exchange with mass spectrometry:

  • Compare exchange patterns of free rplY versus RNA-bound state

  • Identify protected regions representing interaction interfaces

  • Quantify binding-induced conformational changes

These approaches provide residue-level resolution of protein-RNA interactions, enabling detailed mapping of contact points that can be integrated into structural models of the ribosome assembly process .

What strategies can resolve issues with low expression yield of recombinant C. violaceum rplY?

Low expression yield of recombinant rplY can be addressed through systematic troubleshooting:

• Codon optimization:

  • Analyze rare codon usage in the C. violaceum rplY gene

  • Synthesize a codon-optimized version for E. coli

  • Alternatively, use Rosetta strains supplying rare tRNAs

  • This approach typically increases yield by 2-5 fold

• Promoter selection:

  • Test multiple promoter systems (T7, tac, araBAD)

  • Optimize inducer concentration using a gradient approach

  • The T7 promoter system often provides highest yields for ribosomal proteins

• Expression strain screening:

  • Compare expression in multiple E. coli strains (BL21, C41/C43, Arctic Express)

  • Test strains with different protease deficiencies

  • BL21(DE3) pLysS often provides good balance of expression control and yield

• Culture conditions optimization:

  • Test auto-induction media formulations

  • Implement fed-batch cultivation strategies

  • Optimize dissolved oxygen levels during fermentation

  • These approaches can increase yield by 3-10 fold compared to standard batch cultures

Implementing these strategies in combination can increase recombinant rplY yields from typical levels of 10-15 mg/L to 50-100 mg/L in optimized systems.

How can I troubleshoot protein aggregation during purification of C. violaceum rplY?

Protein aggregation during rplY purification can be mitigated through several strategies:

• Buffer optimization:

  • Screen different pH values (pH 6.5-8.5) and buffer systems

  • Test additives that enhance stability:

    • Osmolytes (0.5-1 M glycerol, 0.2-0.5 M sucrose)

    • Salt concentration (150-500 mM NaCl)

    • Reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Include 5-10% glycerol throughout purification

• Solubilizing agents:

  • Add low concentrations of non-denaturing detergents:

    • 0.05-0.1% Triton X-100

    • 0.01-0.05% NP-40

    • 0.1% CHAPS

  • Test arginine (50-100 mM) to prevent aggregation

• Purification strategy modifications:

  • Maintain lower protein concentrations (<2 mg/mL) during initial steps

  • Perform all purification steps at 4°C

  • Implement step-wise dialysis when changing buffer conditions

  • Consider on-column refolding for partially aggregated protein

• Storage optimization:

  • Test protein stability at different concentrations (0.5-5 mg/mL)

  • Add stabilizing excipients for long-term storage

  • Determine optimal storage temperature (-80°C vs. -20°C vs. 4°C)

  • Assess freeze-thaw stability and consider flash-freezing in small aliquots

Implementing these approaches can significantly reduce aggregation, typically improving recovery of monomeric protein by 30-50% during purification .

What approaches can resolve activity loss of recombinant C. violaceum rplY during purification?

Activity loss during purification can be addressed through multiple approaches:

• Cofactor supplementation:

  • Add Mg²⁺ ions (5-10 mM) to all purification buffers

  • Supplement with GTP (0.1-0.5 mM) if needed for proper folding

  • Include trace amounts of 5S rRNA (0.01-0.1 molar ratio) to stabilize native conformation

• Protease inhibition:

  • Use comprehensive protease inhibitor cocktails during cell lysis

  • Include EDTA (1-5 mM) in initial purification steps (remove before IMAC)

  • Keep samples at 4°C and minimize purification duration

• Oxidation prevention:

  • Maintain reducing environment with fresh DTT (1-5 mM)

  • Consider argon or nitrogen sparging of buffers

  • Include antioxidants such as reduced glutathione (1-5 mM)

• Rapid processing:

  • Minimize time between purification steps

  • Use automated chromatography systems if available

  • Process smaller batches to reduce protein exposure time

• Activity validation:

  • Implement activity assays at each purification step

  • Calculate specific activity to track purification efficiency

  • Typical recovery of activity should be >70% through complete purification

Research findings indicate that maintaining Mg²⁺ ions and reducing conditions throughout purification is particularly critical for preserving RNA binding activity of ribosomal proteins .

How can recombinant C. violaceum rplY be used to study bacterial adaptation to environmental stresses?

Recombinant C. violaceum rplY can serve as a model for studying bacterial adaptation mechanisms through the following approaches:

• Stress response studies:

  • Express rplY under various stress conditions (heat shock, oxidative stress)

  • Monitor changes in expression levels and post-translational modifications

  • Correlate structural changes with functional adaptations

  • C. violaceum's adaptation to tropical environments makes its ribosomal proteins particularly suitable for heat stress studies

• Comparative analysis:

  • Compare rplY from C. violaceum with homologs from bacteria adapted to different environments

  • Identify sequence and structural adaptations that correlate with habitat

  • Conduct thermal stability assays across temperature ranges (20-60°C)

• Post-translational modification mapping:

  • Identify stress-induced modifications using mass spectrometry

  • Engineer recombinant rplY variants mimicking these modifications

  • Assess functional impact through RNA binding and ribosome assembly assays

• Structural plasticity assessment:

  • Use hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

  • Compare exchange profiles under different environmental conditions

  • Identify regions with altered flexibility that may contribute to adaptive responses

These approaches provide insights into how ribosomal components contribute to bacterial adaptation to changing environments, with potential implications for understanding pathogenicity mechanisms .

What methodological approaches can assess the impact of rplY mutations on ribosome function?

Several complementary approaches can systematically evaluate how rplY mutations affect ribosome function:

• In vitro translation assays:

  • Reconstitute ribosomes with wild-type or mutant rplY

  • Measure translation efficiency using reporter systems

  • Quantify effects on initiation, elongation, and termination rates

  • Typical results show 20-80% reduction in translation efficiency depending on mutation location

• Ribosome profiling:

  • Express mutant rplY variants in vivo

  • Perform ribosome profiling to assess global translation effects

  • Identify specific mRNAs affected by the mutations

  • Analyze ribosome pause sites and translocation efficiency

• SHAPE analysis (Selective 2'-hydroxyl acylation analyzed by primer extension):

  • Probe rRNA structure in ribosomes containing mutant rplY

  • Map structural changes using next-generation sequencing

  • Correlate structural alterations with functional defects

• Cryo-electron microscopy:

  • Determine structures of ribosomes containing mutant rplY

  • Identify structural perturbations at near-atomic resolution

  • Visualize effects on intersubunit bridges and functional centers

These approaches provide a comprehensive assessment of how specific rplY residues contribute to ribosome structure, assembly, and function, with implications for understanding fundamental aspects of protein synthesis .

How can C. violaceum rplY research contribute to antimicrobial drug development?

Research on C. violaceum rplY can advance antimicrobial drug development through several approaches:

• Structure-based drug design:

  • Identify unique structural features of bacterial rplY proteins

  • Target protein-RNA interfaces specific to bacterial ribosomes

  • Design small molecules that disrupt these specific interactions

  • Virtual screening against rplY binding pockets can identify lead compounds

• High-throughput screening:

  • Develop FRET-based assays to monitor rplY-RNA interactions

  • Screen compound libraries for molecules that disrupt these interactions

  • Implement secondary functional assays to confirm mechanism of action

  • Test promising compounds against clinical pathogen panels

• Peptide inhibitor development:

  • Design peptide mimetics based on ribosomal protein binding interfaces

  • Optimize for cell penetration and stability

  • Test efficacy in cell-based translation assays

  • Assess selectivity for bacterial versus human ribosomes

• Combination therapy approaches:

  • Identify synergistic effects with existing antibiotics

  • Target multiple ribosomal sites simultaneously

  • Evaluate resistance development frequency

This research direction is particularly promising as ribosomal proteins represent evolutionarily conserved targets with essential functions, and exploitation of structural differences between bacterial and eukaryotic ribosomes can lead to selective antimicrobial agents with broad-spectrum activity .