Recombinant 50S ribosomal protein L35 (rpmI)

How should recombinant L35 protein be stored for optimal stability?

According to manufacturer specifications, the stability of recombinant L35 protein depends on several factors:

For reconstitution of lyophilized protein:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (default recommendation is 50%)

• Prepare small aliquots to avoid repeated freeze-thaw cycles, which should be avoided

What expression systems are typically used for recombinant L35 production?

Escherichia coli is the predominant expression system for recombinant L35 protein production . Standard protocols involve:

• Transforming an E. coli expression strain with a plasmid containing the rpmI gene

• Culturing cells in appropriate media (e.g., Luria-Bertani medium with 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl)

• Adding selection antibiotics (e.g., 50 μg/ml kanamycin) to maintain the expression plasmid

• Inducing protein expression with isopropyl β-d-1-thiogalactopyranoside (IPTG) at concentrations around 0.8 mM

• Optimizing expression conditions by testing different:

  • Induction temperatures (17°C, 27°C, or 37°C)

  • Induction times (4, 6, or 8 hours)

  • Culture conditions and agitation rates

How can researchers assess the essentiality of L35 in bacterial survival?

Determining whether L35 is essential for bacterial viability requires a systematic approach:

• Create a conditional mutant strain where:

  • The chromosomal rpmI gene is deleted/replaced with a selectable marker

  • A complementing plasmid carries a functional copy of rpmI under an inducible promoter

  • The plasmid also carries a counter-selectable marker (e.g., sacB)

• Test essentiality through:

  • Growth dependence on inducer: If no growth occurs without inducer (arabinose or IPTG), the gene is essential

  • Plasmid dispensability: Plate cells on media containing 5% sucrose to select for plasmid loss; if no colonies grow, the plasmid (and thus the gene) is essential

• Test temperature sensitivity by repeating tests at different temperatures (e.g., 30°C and 37°C)

In a systematic study of ribosomal protein gene essentiality, 5 ribosomal proteins (L15, L21, L29, L30, and S9) were found to be non-essential, while others were essential for bacterial survival . While L35 wasn't specifically mentioned among the non-essential proteins, using this methodology would conclusively determine its essentiality.

What techniques can overcome the challenges in detecting L35 by mass spectrometry?

As noted in research, "peptides from the shorter ribosomal proteins L31-L36 are more difficult to resolve by [mass spectrometry]" . To improve L35 detection, researchers can employ these strategies:

• Isotope labeling for quantitative comparison:

  • Grow reference strain in 15N-labeled media and experimental strain in 14N media

  • Mix labeled ribosomes in defined ratios (e.g., "14N-labeled ribosomes isolated from our L21 deletion strain, spiked with 15N-labeled ribosomes isolated from the parent DH10B strain")

• Technical improvements:

  • Optimize digestion protocols to generate more detectable L35 peptides

  • Use multiple proteases (beyond standard trypsin) to increase peptide coverage

  • Employ targeted mass spectrometry approaches such as Multiple Reaction Monitoring (MRM)

  • Implement sample fractionation to reduce complexity

  • Consider chemical crosslinking mass spectrometry (XL-MS) to detect L35 through its interaction partners

• Complementary techniques:

  • Western blotting with L35-specific antibodies

  • Ribosome profiling to detect associated mRNAs

  • Fluorescent labeling of L35 for microscopy-based detection

What is the role of L35 in ribosome assembly and biogenesis?

While the specific function of L35 in ribosome assembly isn't explicitly detailed in the available research, principles of ribosomal protein involvement in assembly can be applied:

• Assembly hierarchy: Ribosomal proteins incorporate into ribosomes in a defined order, with early binders facilitating the attachment of later proteins through conformational changes in rRNA

• Assembly checkpoints: Some ribosomal proteins function as quality control checkpoints, with their successful incorporation signaling the readiness for subsequent assembly steps

• Interaction networks: Research indicates that ribosomal proteins form critical interactions "with functional sites of the ribosome," and "the importance of these interactions is poorly understood, in part due to the lack of a facile experimental system to create and analyze mutations in ribosomal proteins"

• Late-stage assembly: Some ribosomal proteins participate in late steps of 50S ribosomal subunit assembly, with specific modifications triggering conformational changes that facilitate maturation, as demonstrated with other ribosomal components: "Um2552 formation by RlmE facilitates interdomain interactions between H92 and H71, in concert with the incorporation of L36 and other ribosomal proteins"

To specifically study L35's role in assembly, researchers could:

• Monitor assembly intermediates that accumulate in L35 depletion conditions

• Analyze the timing of L35 incorporation using pulse-chase experiments

• Perform in vitro reconstitution experiments with and without L35

How can researchers use L35 truncation studies to understand its functional domains?

Based on successful approaches with other ribosomal proteins, researchers can use truncation studies to identify functional domains within L35:

• Design truncation strategy:

  • Create a series of N-terminal and C-terminal truncations of L35

  • Express truncated variants from a plasmid in a strain with chromosomal L35 deletion

  • Ensure proper expression using an inducible promoter system (e.g., trc promoter)

• Functional assessment:

  • Test growth complementation by each truncation variant

  • Evaluate ribosome assembly by sedimentation analysis

  • Assess translation efficiency using reporter systems

• Incorporation analysis:

  • Use quantitative mass spectrometry to determine if truncated variants incorporate into ribosomes

  • As demonstrated with other ribosomal proteins: "Mutated or truncated ribosomal protein variants may have altered affinities for the ribosome... which may imply a reduced interaction with the ribosome"

This approach successfully identified functional domains in other ribosomal proteins: "In the 50S P site, removal of the L5 loop interacting with tRNA (L5Δ70-83) caused a severe growth defect" while "truncation of 9 amino acids from the N-terminal tail of L27 (L27NΔ9)... minimally affected cell growth" .

What in vitro methods can be used to study L35's role in ribosome assembly?

Researchers can establish an in vitro system to study L35's role in ribosome assembly:

• Ribosome reconstitution approach:

  • "Each subunit can be reconstituted in vitro from its individual component ribosomal RNAs and proteins, indicating that these components contain all of the information necessary to automatically assemble into functional subunits"

  • Compare assembly kinetics and efficiency with and without L35

  • Monitor assembly intermediates using sedimentation analysis

• Assembly conversion assays:

  • Isolate assembly intermediates from L35-depleted cells

  • Attempt to convert these to mature 50S subunits by adding purified L35

  • Similar approaches have been successful for other assembly factors: "We partially recapitulate the formation of 50S from 45S in the presence of recombinant RlmE and AdoMet in vitro"

• Tracking assembly using density gradients:

  • Use sucrose density gradient (SDG) analysis at different Mg2+ concentrations to monitor structural transitions

  • "In SDG analysis performed at 0.5 mM Mg2+, the level of 45S particle gradually decreased, and about half of the particle was converted to 50S within 120 min"

• Pulse-chase analysis:

  • Label rRNA with isotopes (e.g., 13C-Ado)

  • Track the movement of labeled rRNA through assembly intermediates to mature subunits

  • "The proportion of 13C-labeled rRNAs in 45S plunged during the first 80 min" while "13C-labeled rRNAs in 50S clearly increased"

What quality control methods should be applied to recombinant L35 preparations?

To ensure the integrity and functionality of recombinant L35 preparations:

• Purity assessment:

  • SDS-PAGE analysis (commercial preparations typically achieve >85% purity)

  • Mass spectrometry to confirm protein identity and detect potential modifications

  • Size exclusion chromatography to verify homogeneity

• Functional testing:

  • RNA binding assays to confirm interaction with rRNA

  • In vitro reconstitution to verify incorporation into ribosomal subunits

  • Circular dichroism spectroscopy to assess secondary structure

• Stability monitoring:

  • Thermal shift assays to determine melting temperature

  • Time-course analysis of protein under various storage conditions

  • Aggregation assessment using dynamic light scattering

These quality control measures ensure that experimental outcomes truly reflect L35 biology rather than artifacts from compromised protein preparations.

How can researchers optimize L35 expression to increase yield and solubility?

Based on methods used for recombinant protein production in bacterial systems:

• Strain selection and culture optimization:

  • Test multiple E. coli strains designed for protein expression

  • Optimize media composition (defined vs. complex media)

  • Implement fed-batch or high-density culture techniques

• Expression conditions:

  • Systematically vary induction parameters:

    • IPTG concentration (typically around 0.8 mM)

    • Temperature (17°C, 27°C, and 37°C)

    • Induction duration (4, 6, and 8 hours)

• Construct engineering:

  • Add solubility-enhancing tags (e.g., SUMO, MBP, or GST)

  • Codon-optimize the rpmI sequence for the expression host

  • Co-express molecular chaperones to facilitate folding

• Special culture systems:

  • Consider specialized culture vessels: "The cell suspension was diluted (1:10) in two HARV vessels filled with fresh LB medium containing 50 μg/ml kanamycin"

  • Test different agitation rates: "The SMG culture process was carried out under different rotary speeds (10, 15, 20, and 30 rpm)"

A systematic optimization approach, testing these variables individually and in combination, will likely yield significant improvements in recombinant L35 production.

How can purified L35 be used to study ribosome assembly defects?

Purified recombinant L35 can be a valuable tool for studying ribosome assembly:

• Complementation studies:

  • Add purified L35 to assembly-defective ribosomes from L35-depleted cells

  • Monitor conversion of precursor particles to mature subunits through sedimentation analysis

  • Similar approaches have demonstrated: "The 50S peak slightly increased, concomitant with a decrease in the 45S peak, only in the presence of both RlmE and AdoMet"

• Interaction partner identification:

  • Use purified L35 as bait in pull-down assays to identify binding partners

  • Perform cross-linking studies to capture transient interactions

  • Map the L35 interaction network within the ribosome

• In vitro assembly systems:

  • Establish defined reconstitution systems with purified components

  • Systematically test the order of addition to determine L35's position in the assembly hierarchy

  • Modify L35 (mutations, truncations) to identify critical functional regions

• Assembly kinetics:

  • Use fluorescently labeled L35 to monitor incorporation rates

  • Perform stopped-flow kinetics to measure binding rates to assembly intermediates

  • Compare wild-type versus modified L35 to identify rate-limiting steps

What insights does L35 provide into bacterial ribosome evolution?

Although not explicitly covered in the search results, L35's evolutionary significance can be inferred:

• Comparative genomics:

  • Analyze L35 sequences across bacterial phyla to identify conserved and variable regions

  • Map conservation patterns onto structural models to identify functionally important domains

  • Compare with archaeal and eukaryotic homologs (if present) to understand ribosome evolution

• Structure-function relationships:

  • Determine whether L35 occupies similar positions within ribosomes from diverse bacterial species

  • Analyze co-evolution patterns between L35 and its rRNA binding regions

  • Identify adaptive changes in L35 that correlate with environmental niches of different bacteria

• Essentiality patterns:

  • Compare the essentiality of L35 across different bacterial species

  • Correlate with genome size, growth rate, and ecological niche

  • Research has shown varying essentiality patterns for other ribosomal proteins: "5 mutants (with chromosomal deletion of the gene encoding L15, L21, L29, L30, or S9) showed obvious growth in the absence of an inducer"

This evolutionary perspective can guide rational antibiotic design targeting bacterial-specific features of the ribosome, including potential L35 interactions.

What emerging technologies could advance our understanding of L35 function?

Several cutting-edge approaches could significantly enhance our understanding of L35:

• Cryo-electron microscopy:

  • Capture high-resolution structures of ribosomes with and without L35

  • Visualize different conformational states during assembly

  • Map L35's position relative to functional centers of the ribosome

• Time-resolved structural methods:

  • Time-resolved cryo-EM to capture assembly intermediates

  • Single-molecule FRET to monitor L35 incorporation dynamics

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

• Genomic approaches:

  • CRISPR interference for titratable control of L35 expression

  • Ribosome profiling to assess translation effects of L35 depletion

  • Synthetic genetic arrays to identify genetic interactions with L35

• Computational methods:

  • Molecular dynamics simulations of L35-rRNA interactions

  • Quantum mechanics/molecular mechanics studies of L35's role in catalytic centers

  • Systems biology models integrating L35 into ribosome assembly pathways

These approaches, used in combination, would provide multidimensional insights into L35 function beyond what's possible with current methodologies.