Recombinant Mannheimia succiniciproducens 50S ribosomal protein L6 (rplF)

What expression systems are recommended for producing recombinant M. succiniciproducens ribosomal protein L6?

For recombinant production of M. succiniciproducens ribosomal protein L6, E. coli-based expression systems are generally recommended due to their well-established protocols and high yield potential. Prokaryotic expression systems using E. coli BL21(DE3) strains have been successfully employed for producing recombinant proteins, including ribosomal components . The methodology typically involves cloning the rplF gene into an expression vector with an appropriate promoter (such as T7) and affinity tag (commonly N-terminal His-tag for easier purification) . When designing the expression construct, it is advisable to include tag sequences that facilitate downstream purification without interfering with protein folding. The expression conditions should be optimized to balance between high yield and proper folding, as ribosomal proteins may form inclusion bodies when overexpressed .

What purification methods are most effective for isolating recombinant M. succiniciproducens L6 protein?

The most effective purification strategy for recombinant M. succiniciproducens L6 protein typically employs immobilized metal affinity chromatography (IMAC) using Ni-nitrilotriacetic acid (Ni-NTA) columns when the protein is expressed with a histidine tag . The general purification protocol includes:

• Cell lysis under native or denaturing conditions depending on protein solubility

• Initial capture using Ni-NTA affinity chromatography

• Washing steps with increasing imidazole concentrations to remove non-specifically bound proteins

• Elution with high imidazole concentration buffer

• Further purification using size exclusion chromatography if higher purity is required

For optimal results, buffer conditions should be carefully adjusted to maintain protein stability. A typical buffer formulation might include PBS at pH 7.4 with stabilizing agents such as trehalose . The purified protein can then be analyzed by SDS-PAGE to confirm size and purity, with expected molecular mass potentially differing from predicted values due to factors like post-translational modifications or the relative charge of amino acids .

How can I verify the identity and activity of purified recombinant M. succiniciproducens L6 protein?

Verification of recombinant M. succiniciproducens L6 protein identity and activity requires multiple analytical approaches:

For functional verification, a complementation assay using L6-depleted bacterial strains can be particularly informative. In such assays, the recombinant L6 should restore normal growth patterns and ribosomal activity when introduced into L6-deficient cells . Additionally, in vitro ribosome reconstitution experiments can demonstrate the ability of purified L6 to incorporate into ribosomal subunits and contribute to translation activity.

What are the structural differences between M. succiniciproducens L6 and its homologs in other bacterial species?

Comparative structural analysis of M. succiniciproducens L6 with homologs from other bacterial species requires detailed examination of amino acid sequences, secondary structure elements, and tertiary folding patterns. While specific structural data for M. succiniciproducens L6 is limited in the available search results, comparison can be made with related Pasteurellaceae family members like Mannheimia haemolytica, which shares genomic similarities .

The approach to determining structural differences includes:

• Multiple sequence alignment of L6 proteins from M. succiniciproducens and other bacterial species

• Homology modeling based on crystal structures of L6 from model organisms

• Analysis of conserved domains and variable regions

• Examination of amino acid substitutions at key functional sites

Based on genomic analysis of related Pasteurellaceae species, M. succiniciproducens likely forms a distinct lineage with Actinobacillus pleuropneumoniae and Haemophilus ducreyi . This phylogenetic relationship suggests that the L6 protein may contain unique structural features compared to more distant bacterial species. These differences might be concentrated in surface-exposed regions while maintaining conservation in RNA-binding domains essential for ribosomal function.

How does the absence of L6 affect ribosome assembly and function in bacterial systems?

The absence of L6 protein has profound effects on ribosome assembly and function, as demonstrated in E. coli studies that provide insights applicable to M. succiniciproducens. When L6 is depleted, bacteria exhibit a characteristic biphasic growth pattern: initial limited growth (LI-phase), followed by a suspension period (S-phase), and eventual resumption of growth (LII-phase) . This growth pattern corresponds to specific defects in ribosome assembly:

• Accumulation of 50S subunit precursors (45S particles) that completely lack L6

• Reduced factor-dependent GTPase activity in the ribosomes

• Defects in late-stage assembly of the 50S ribosomal subunit

The molecular basis for these effects likely stems from L6's position near the sarcin/ricin loop, which is crucial for interaction with GTPase translation factors. Without L6, the ribosome structure is compromised in ways that prevent normal translation factor binding and activity. The eventual resumption of growth in L6-depleted cells has been attributed to leaky expression of L6 from complementing plasmids in experimental systems, suggesting that even small amounts of L6 can support minimal ribosomal function .

What challenges might be encountered when expressing recombinant M. succiniciproducens L6, and how can they be addressed?

Expression of recombinant M. succiniciproducens L6 presents several potential challenges that require specific methodological solutions:

One of the most significant challenges is inclusion body formation, a common occurrence with recombinant protein expression in E. coli . For ribosomal proteins like L6, which normally exist in complex with rRNA, expression without their natural binding partners can lead to misfolding and aggregation. Temperature reduction during induction (from 37°C to 16-25°C) and decreasing inducer concentration can promote proper folding. Alternatively, if inclusion bodies persist, established protocols for solubilization and refolding can be implemented, typically involving denaturants like urea or guanidine hydrochloride followed by controlled dialysis .

How can we design experimental approaches to investigate the role of M. succiniciproducens L6 in antibiotic resistance mechanisms?

Investigating the role of M. succiniciproducens L6 in antibiotic resistance requires multifaceted experimental approaches that target different aspects of ribosome-antibiotic interactions:

• Structure-function analyses:

  • Site-directed mutagenesis of conserved residues in L6

  • Complementation studies in L6-depleted strains with mutant variants

  • Measurement of antibiotic binding affinities to ribosomes containing wild-type vs. mutant L6

• In vitro translation assays:

  • Reconstitution of ribosomes with and without L6

  • Assessment of translation efficiency in the presence of various antibiotics

  • Determination of IC50 values for different antibiotics with various L6 mutants

• In vivo approaches:

  • Generation of M. succiniciproducens strains with chromosomal L6 mutations

  • Growth inhibition assays in the presence of ribosome-targeting antibiotics

  • Selection of spontaneous antibiotic-resistant mutants and sequencing of L6 gene

Since L6 is positioned near functional centers of the ribosome, including the sarcin/ricin loop that interacts with translation factors , mutations in L6 might affect binding of antibiotics that target these regions. Comparative studies with related bacterial species can provide insights into the conservation of resistance mechanisms. The experimental design should include appropriate controls, such as testing antibiotics known to target regions distant from L6 as negative controls.

What are the implications of L6 protein variations across Mannheimia species for evolutionary studies and taxonomic classification?

The variations in L6 protein across Mannheimia species offer valuable insights for evolutionary studies and taxonomic classification within the Pasteurellaceae family. Comparative genomic analyses have shown that Mannheimia haemolytica, a related species to M. succiniciproducens, forms a lineage with Actinobacillus pleuropneumoniae and Haemophilus ducreyi that is distinct from other Pasteurellaceae . This phylogenetic relationship, supported by analysis of housekeeping genes, suggests that L6 protein sequences could serve as molecular markers for evolutionary studies.

Research approaches to explore these implications include:

• Comprehensive phylogenetic analysis using L6 sequences from all available Mannheimia species

• Calculation of synonymous vs. non-synonymous substitution rates to identify selective pressures

• Identification of signature residues that distinguish Mannheimia from other Pasteurellaceae

• Correlation of L6 variations with ecological niches and host specificity

The genome of M. haemolytica includes 2,839 coding sequences, with 1,966 assigned functions and 436 being unique to this species . Similar genomic analysis of M. succiniciproducens focusing on the rplF gene (encoding L6) could reveal species-specific adaptations. The high conservation of ribosomal proteins makes variations particularly significant, potentially reflecting adaptive responses to different environmental pressures or host interactions.

What protocols are recommended for site-directed mutagenesis of M. succiniciproducens L6 to study structure-function relationships?

For site-directed mutagenesis of M. succiniciproducens L6, the following methodological approach is recommended:

• Target selection:

  • Identify conserved residues through multiple sequence alignment

  • Focus on residues near the rRNA binding site or at the interface with other ribosomal components

  • Select residues based on structural predictions or homology models

• Mutagenesis protocol:

  • Design primers with desired mutations following the QuikChange methodology

  • Perform PCR amplification using high-fidelity polymerase

  • Digest parental DNA with DpnI

  • Transform competent E. coli cells with the reaction product

• Verification steps:

  • Sequence the entire rplF gene to confirm the desired mutation and absence of unwanted changes

  • Express and purify the mutant protein following standard protocols

  • Verify correct folding through circular dichroism or limited proteolysis

• Functional analysis:

  • Assess binding to 23S rRNA through electrophoretic mobility shift assays

  • Test incorporation into 50S ribosomal subunits

  • Evaluate effects on ribosome assembly and translation activity

For comprehensive structure-function studies, a systematic alanine scanning approach can be employed, where conserved residues are sequentially replaced with alanine to identify essential amino acids. Additionally, conservative and non-conservative substitutions can provide insights into the physicochemical requirements at specific positions.

How can we develop a reconstitution system to study the role of M. succiniciproducens L6 in ribosome assembly in vitro?

Developing an in vitro reconstitution system for studying M. succiniciproducens L6 in ribosome assembly requires a systematic approach that builds upon established methodologies for bacterial ribosome reconstitution:

• Component preparation:

  • Express and purify recombinant L6 protein using IMAC and size exclusion chromatography

  • Isolate total rRNA from M. succiniciproducens or prepare synthetic 23S rRNA

  • Express and purify other essential ribosomal proteins

• Assembly protocol development:

  • Adapt established E. coli ribosome reconstitution protocols

  • Determine optimal buffer conditions (Mg²⁺ concentration, ionic strength, pH)

  • Establish a sequential addition protocol based on assembly hierarchy

• Validation methods:

  • Sucrose gradient centrifugation to analyze formation of ribosomal particles

  • Negative-stain electron microscopy to visualize assembled structures

  • Functional assays such as peptidyl transferase activity measurement

• L6-specific investigations:

  • Compare assembly with and without L6

  • Use fluorescently labeled L6 to monitor incorporation kinetics

  • Test L6 mutants to identify critical regions for assembly

The experimental design should include appropriate controls, such as reconstitution with E. coli L6 to assess cross-species functionality. A particular focus should be placed on the formation of 45S particles, which have been identified as precursors in L6-depleted conditions , to understand the precise stage at which L6 acts during assembly.

What are the best approaches for comparing the interactions of wild-type versus modified M. succiniciproducens L6 with ribosomal RNA?

To compare interactions between wild-type and modified M. succiniciproducens L6 proteins with ribosomal RNA, several complementary approaches can be employed:

The experimental workflow should begin with expression and purification of wild-type and modified L6 proteins under identical conditions to ensure comparable quality. For RNA preparation, either synthetic oligonucleotides corresponding to helix 97 of 23S rRNA (the primary binding site of L6) or full-length 23S rRNA can be used, depending on the specific question being addressed.

For detailed mapping of interaction sites, hydroxyl radical footprinting or selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) can identify nucleotides protected by L6 binding or conformational changes induced in the RNA. These approaches can reveal subtle differences in how modifications to L6 affect its interaction with rRNA.

How can we establish a system to study the impact of M. succiniciproducens L6 on ribosome function in vivo?

Establishing a system to study M. succiniciproducens L6 function in vivo requires development of genetic tools and assay systems:

• Construction of conditional expression strain:

  • Generate an M. succiniciproducens strain with chromosomal rplF gene disruption

  • Complement with plasmid-borne rplF under control of an inducible promoter

  • Verify arabinose-dependent growth pattern similar to that observed in E. coli L6 depletion studies

• Development of reporter systems:

  • Integrate translational reporter constructs (GFP, luciferase) to monitor translation efficiency

  • Design reporters with programmed frameshifting or stop codon readthrough to assess translation fidelity

  • Introduce translation-dependent antibiotic resistance markers for selection-based screens

• Methods for ribosome isolation and analysis:

  • Establish protocols for efficient lysis of M. succiniciproducens cells

  • Optimize sucrose gradient conditions for separation of ribosomal subunits

  • Develop quantitative mass spectrometry approaches to assess ribosome composition

• Phenotypic characterization approaches:

  • Growth curve analysis under various stress conditions

  • Antibiotic susceptibility testing

  • Metabolic profiling to detect changes in cellular physiology

If direct genetic manipulation of M. succiniciproducens proves challenging, an alternative approach is to use E. coli as a surrogate host, creating a strain with its endogenous rplF replaced by the M. succiniciproducens ortholog. This approach has been successfully employed for studying other ribosomal proteins and would allow utilization of the well-established E. coli genetic toolbox.

What strategies can be employed when recombinant M. succiniciproducens L6 forms inclusion bodies during expression?

When recombinant M. succiniciproducens L6 forms inclusion bodies during expression, several strategies can be implemented for optimization:

• Prevention strategies:

  • Reduce expression temperature to 16-20°C to slow protein synthesis and facilitate proper folding

  • Decrease inducer concentration to reduce expression rate

  • Use rich media supplemented with rare codon tRNAs if codon usage is an issue

  • Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist folding

  • Fuse L6 to solubility-enhancing tags such as MBP, SUMO, or Thioredoxin

• Recovery strategies (if prevention fails):

  • Solubilize inclusion bodies using denaturing agents (8M urea or 6M guanidine hydrochloride)

  • Perform refolding through gradual removal of denaturant via dialysis or dilution

  • Test various refolding additives (L-arginine, glycerol, sucrose) to prevent aggregation

  • Consider on-column refolding during affinity purification

• Alternative expression systems:

  • Test different E. coli expression strains (Origami for disulfide bond formation, Arctic Express for low-temperature expression)

  • Consider cell-free protein synthesis systems which can produce soluble protein by avoiding cellular constraints

The formation of inclusion bodies is a common challenge when expressing recombinant proteins in E. coli . For ribosomal proteins like L6, which naturally exist in complex with rRNA and other proteins, expressing them in isolation can lead to improper folding and aggregation. Systematic optimization of expression conditions, combined with appropriate fusion partners and folding aids, can significantly improve soluble yield.

How should experimental conditions be adjusted when studying interactions between M. succiniciproducens L6 and ribosomes from different bacterial species?

When studying interactions between M. succiniciproducens L6 and ribosomes from different bacterial species, several experimental adjustments are necessary to account for species-specific differences:

• Buffer optimization:

  • Adjust ionic conditions to match the physiological environment of each species

  • Test multiple Mg²⁺ concentrations (typically 5-20 mM) as ribosome stability varies across species

  • Consider adding polyamines (spermidine, spermine) which can stabilize heterologous interactions

• Cross-species binding assessment:

  • Perform comparative binding assays (EMSA, filter binding) with L6 from multiple species

  • Use competition assays to determine relative binding affinities

  • Label L6 with fluorescent tags to directly visualize incorporation into heterologous ribosomes

• Functional characterization:

  • Develop hybrid in vitro translation systems containing native ribosomes with heterologous L6

  • Evaluate translation efficiency and fidelity using reporter mRNAs

  • Test response to antibiotics that target the L6-adjacent regions

• Structural considerations:

  • Generate homology models of M. succiniciproducens L6 based on known structures

  • Identify potential interface residues that might affect cross-species compatibility

  • Consider the co-evolution of L6 with its rRNA binding site

Species within the Pasteurellaceae family, such as M. haemolytica and A. pleuropneumoniae, share genomic similarities with M. succiniciproducens , suggesting their ribosomal components may be more compatible than those from distant bacterial families. Experimental controls should include homologous systems (L6 with its native ribosome) to establish baseline interaction parameters.

What are effective strategies for optimizing the yield and purity of recombinant M. succiniciproducens L6 protein?

Optimizing yield and purity of recombinant M. succiniciproducens L6 protein requires a comprehensive approach addressing multiple aspects of the expression and purification process:

• Expression optimization:

  • Test multiple E. coli expression strains (BL21(DE3), Rosetta, C41/C43)

  • Evaluate different induction parameters (OD600 at induction, inducer concentration, temperature)

  • Compare various media formulations (LB, TB, auto-induction media)

  • Conduct small-scale expression trials with different construct designs

• Construct optimization:

  • Test N-terminal vs. C-terminal affinity tags

  • Evaluate different tag options (His, GST, MBP) for solubility and purification efficiency

  • Include or exclude linker sequences between tag and protein

  • Consider codon optimization for E. coli expression

• Purification refinement:

  • Develop optimized cell lysis protocols (sonication parameters, lysis buffer composition)

  • Fine-tune IMAC conditions (imidazole gradient, flow rate, column volume)

  • Implement secondary purification steps (ion exchange, size exclusion chromatography)

  • Test various buffer systems for improved stability during purification

• Quality assessment:

  • Regular SDS-PAGE analysis at each purification stage

  • Consider dynamic light scattering to assess aggregation state

  • Verify activity through functional assays (RNA binding, incorporation into ribosomes)

Expected yields for well-optimized ribosomal protein expression systems typically range from 5-20 mg/L of culture, with purity exceeding 90% after two-step purification . The purified protein should be stored in stabilizing buffer conditions, potentially including trehalose or glycerol to prevent aggregation during storage .

How can we address the challenge of ribosomal protein L6 instability during purification and storage?

Addressing L6 protein instability during purification and storage requires implementing multiple stabilization strategies:

• Buffer optimization:

  • Screen various pH conditions (typically pH 6.5-8.0) to identify optimal stability range

  • Test different ionic strengths (100-500 mM NaCl or KCl)

  • Include stabilizing agents such as trehalose (5-10%) , glycerol (5-20%), or sucrose (5-10%)

  • Add reducing agents (DTT, β-mercaptoethanol) if cysteine residues are present

• Handling protocols:

  • Maintain samples at 4°C during purification

  • Avoid freeze-thaw cycles by aliquoting purified protein

  • Consider flash-freezing in liquid nitrogen before storage at -80°C

  • Use low-protein-binding tubes and filtration membranes

• Co-factor considerations:

  • Test addition of nucleic acid fragments (RNA oligonucleotides corresponding to binding sites)

  • Evaluate divalent cations (Mg²⁺, Mn²⁺) that might stabilize protein conformations

  • Consider molecular crowding agents (PEG, Ficoll) to mimic cellular environment

• Storage formulation:

  • Prepare as freeze-dried powder for maximum stability

  • For solution storage, concentrate to 0.5-5 mg/mL range

  • Filter sterilize (0.22 μm) to remove potential nucleation sites for aggregation

  • Consider addition of non-ionic detergents (0.01% NP-40 or Triton X-100) for highly hydrophobic variants

As ribosomal proteins like L6 naturally exist in complex with rRNA, they may exhibit instability when purified as individual components. One effective approach is to include RNA fragments corresponding to the natural binding site (helix 97 of 23S rRNA for L6) in the storage buffer, which can stabilize the protein by satisfying its RNA-binding interface.