Recombinant Gloeobacter violaceus 50S ribosomal protein L14 (rplN)

What is the genomic context of the rplN gene in Gloeobacter violaceus?

The rplN gene in G. violaceus is part of its fully sequenced genome. G. violaceus PCC 7421 has a circular chromosome of approximately 4.66 Mbp . Unlike some genes in G. violaceus (such as the rhodopsin gene which is localized alone in the genome), ribosomal protein genes are typically clustered in operons . For genomic analysis of rplN, researchers should employ comparative genomic approaches similar to those used in pangenomic analyses of Gloeobacter species, which have revealed unique gene clusters among different species .

What expression systems are most effective for producing recombinant G. violaceus rplN?

Based on successful expression of other G. violaceus proteins, Escherichia coli is recommended as the primary expression system for recombinant rplN. Specifically, construct a vector containing the rplN gene with a 6×His-tag for purification purposes. For optimal expression, use BL21(DE3) E. coli strains with IPTG induction, similar to methods used for Gloeorhodopsin expression . Expression should be confirmed via SDS-PAGE and Western blot analysis using anti-His antibodies at a 1:6,000 dilution with HRP-conjugated secondary antibodies at 1:15,000 dilution .

How can I optimize purification of recombinant G. violaceus rplN?

Purification of recombinant rplN should utilize affinity chromatography with Ni²⁺-NTA resin if a His-tag is incorporated into the construct. After initial purification, researchers should perform size-exclusion chromatography to enhance protein purity. Protein concentration can be determined using a Bio-Rad Dc protein assay kit . For structural studies, additional purification steps may be necessary, including ion-exchange chromatography to remove contaminants that co-purify with the target protein.

How does G. violaceus rplN differ structurally from other cyanobacterial ribosomal L14 proteins?

Structural comparison requires computational modeling approaches similar to those used for other G. violaceus proteins. To analyze structural differences:

• Generate sequence alignments using ClustalX with ribosomal L14 sequences from different cyanobacteria

• Adjust alignments manually based on secondary structure predictions using psipred

• Employ comparative modeling using available crystal structures as templates

• Run multiple modeling trajectories (15,000-20,000) against each template structure

• Cluster models based on RMSD and evaluate top clusters based on total-score

This approach, similar to the methodology used for studying AtpD and AtpG proteins , will reveal structural conservation and divergence in the rplN protein across cyanobacterial lineages.

What are the optimal conditions for functional assays of recombinant G. violaceus rplN?

Functional assays for recombinant rplN should evaluate its ability to incorporate into ribosomes and support protein synthesis. Researchers should:

• Perform in vitro ribosome reconstitution assays using purified rplN and other ribosomal components

• Test different buffer conditions (pH 6.5-8.0, Mg²⁺ concentrations 5-20 mM)

• Evaluate ribosome assembly using sucrose gradient ultracentrifugation

• Measure translation efficiency with in vitro translation systems

The protein is likely stable at physiological pH (7.4) based on observations of other G. violaceus proteins , but researchers should empirically determine optimal buffer conditions for maximum function.

How do site-directed mutations affect G. violaceus rplN structure and function?

Site-directed mutagenesis can be performed using the two-step megaprimer PCR method with Pfu polymerase as demonstrated for Gloeorhodopsin . Target conserved residues identified through sequence alignment with homologous proteins. After generating mutants, compare:

• Secondary structure changes using circular dichroism spectroscopy

• Thermal stability differences using differential scanning fluorimetry

• Ribosome incorporation efficiency through reconstitution assays

• Effects on translation fidelity and rate using in vitro translation systems

Results should be analyzed in the context of the protein's predicted structure to understand structure-function relationships.

How can recombinant G. violaceus rplN be used to study evolutionary relationships among cyanobacteria?

Evolutionary analyses using rplN should compare sequence and structural conservation across diverse cyanobacterial lineages. Methodology includes:

• Collect rplN sequences from various cyanobacteria including primitive genera like Gloeobacter and Aurora

• Generate multiple sequence alignments and phylogenetic trees using maximum likelihood methods

• Calculate sequence conservation indices for functional domains

• Compare with phylogenies based on other markers like 16S rRNA

This approach will help determine if rplN evolution mirrors organismal evolution. Recent pangenomic analyses of Gloeobacter species revealed that despite G. morelensis and G. violaceus sharing 99.93% 16S rRNA gene identity, they have only 92.6% average nucleotide identity (ANI) . Similar detailed comparisons of rplN could provide insights into ribosomal protein evolution in cyanobacteria.

What techniques are most effective for studying interactions between rplN and other ribosomal components?

To study interactions between recombinant rplN and other ribosomal components:

• Use pull-down assays with tagged rplN to identify binding partners

• Employ hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Perform chemical cross-linking followed by mass spectrometry (XL-MS) to identify proximal residues

• Apply cryo-electron microscopy to visualize rplN within the assembled ribosome

For in vivo interaction studies, consider generating antibodies against purified rplN using the protocol described for Gloeorhodopsin antibody production , and perform co-immunoprecipitation experiments.

How does the unique evolutionary position of Gloeobacter affect rplN structure and function compared to other bacteria?

Given that Gloeobacter is considered one of the most primitive extant cyanobacteria that lacks thylakoids , its rplN may retain ancestral features. To investigate:

• Compare ribosome assembly mechanisms between Gloeobacter and other cyanobacteria

• Analyze the rate of evolutionary change in rplN across bacterial lineages

• Test functional interchangeability by substituting rplN in heterologous systems

• Examine coevolution patterns between rplN and interacting ribosomal components

What is the optimal experimental design for expressing and purifying G. violaceus rplN for structural studies?

For structural studies, design an expression construct with:

• N-terminal His-tag with a TEV protease cleavage site

• Codon optimization for E. coli expression

• Auto-induction media for high-density cultures

• Lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol

Purification should include:

• Ni²⁺-NTA affinity chromatography

• TEV protease cleavage

• Reverse Ni²⁺-NTA chromatography

• Size-exclusion chromatography

• Concentration to 10-15 mg/mL for crystallization trials

Protein purity should be monitored by SDS-PAGE and western blotting using procedures established for other G. violaceus proteins .

How can transcriptomic approaches be integrated to study rplN expression in different growth conditions?

To study rplN expression patterns:

• Culture G. violaceus under varying conditions (light intensity, temperature, nutrient availability)

• Extract total RNA using Trizol-based methods as demonstrated for G. violaceus genomic DNA extraction

• Perform RNA-Seq with stranded library preparation

• Map reads to the G. violaceus genome using Bowtie2

• Calculate RPKM values for rplN and other genes using Artemis

• Visualize transcript expression profiles using Artemis and/or IGV

Compare expression patterns with other ribosomal proteins and correlate with growth rates to understand regulation of ribosome biogenesis in this primitive cyanobacterium.

What are common challenges in expressing soluble G. violaceus rplN and how can they be addressed?

Common challenges include:

• Inclusion body formation: Modify expression conditions by reducing temperature to 18°C and IPTG concentration to 0.1 mM

• Protein instability: Add stabilizing agents (glycerol 10-15%, reducing agents)

• Low expression levels: Test different E. coli strains (BL21, Rosetta, Arctic Express)

• Co-purification contaminants: Implement more stringent washing during affinity purification

If expression in E. coli fails, consider alternative systems such as cell-free protein synthesis or yeast expression systems with appropriate modifications.

How can researchers address discrepancies in functional assays between recombinant and native rplN?

To address functional discrepancies:

• Compare post-translational modifications between native and recombinant proteins

• Validate folding using circular dichroism and fluorescence spectroscopy

• Test different buffer compositions mimicking the cytoplasmic environment of G. violaceus

• Express the protein with different tags and compare activity

• Consider co-expression with chaperones or partner proteins

Additionally, validate results by purifying native rplN directly from G. violaceus cultures for comparative analyses.