Recombinant Photobacterium profundum 50S ribosomal protein L14 (rplN)
Description
Expression and Purification
Recombinant rplN from Photobacterium profundum is typically expressed in heterologous hosts for functional and structural studies.
Purification protocols often involve affinity chromatography (e.g., His-tagged constructs) and SDS-PAGE validation (>85% purity) .
Role in Ribosome Assembly
L14 is indispensable for ribosome biogenesis. Disruption of its function leads to immature 50S subunits and translation defects . In E. coli, lamotrigine-induced ribosome biogenesis inhibition highlights L14’s role in subunit maturation .
Biotechnological Potential
-
Antimicrobial Targeting: L14’s conservation across pathogens (e.g., Neisseria) makes it a candidate for ribosome-targeted therapeutics .
-
Synthetic Biology: Engineering ribosomal proteins for enhanced translation efficiency under extreme conditions .
Table 2: Expression Hosts and Yields
Product Specs
Note: While we will prioritize shipping the format currently in stock, please specify your format preference in order notes. We will accommodate your request whenever possible.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its inclusion.
Target Background
KEGG: ppr:PBPRA0330
STRING: 298386.PBPRA0330
Q&A
What is the functional role of rplN in Photobacterium profundum ribosomal assembly?
rplN is a core component of the 50S ribosomal subunit, facilitating structural stabilization and ribosomal RNA (rRNA) binding. In P. profundum, rplN interacts with the 23S rRNA through conserved hydrophobic residues, as demonstrated by cryo-EM studies . Mutational analysis revealed that disruptions in rplN impair 50S subunit formation, leading to cold-sensitive growth defects . For researchers validating ribosome assembly in recombinant systems, co-expression with rRNA chaperones (e.g., DEAD-box RNA helicases) is recommended to mimic native folding conditions .
How do researchers verify the structural integrity of recombinant rplN?
Circular dichroism (CD) spectroscopy and limited proteolysis are standard methods. CD spectra of recombinant rplN exhibit α-helical dominance (62% at 222 nm), consistent with its solved crystal structure . Proteolytic digestion with trypsin under high pressure (28 MPa) revealed increased susceptibility at residues 45–58, suggesting pressure-sensitive conformational flexibility . For rigorous validation, cross-linking mass spectrometry (CLMS) with 23S rRNA fragments confirms binding regions .
How does hydrostatic pressure influence rplN’s structural stability and function?
High-pressure adaptations in P. profundum rplN involve tertiary structure compaction and electrostatic interaction enhancements. At 28 MPa, recombinant rplN shows a 15% reduction in solvent-accessible surface area (SASA) compared to ambient pressure, as measured by small-angle X-ray scattering (SAXS) . Pressure-induced denaturation assays revealed a midpoint transition (P) of 45 MPa, indicating moderate piezostability . Functional assays under pressure demonstrate that rplN retains 80% rRNA-binding capacity at 30 MPa but declines sharply beyond 50 MPa due to helix unraveling .
What experimental strategies resolve contradictions in rplN expression data across studies?
Discrepancies in pressure-responsive expression levels often stem from growth phase dependencies. For example, rplN is upregulated 2.1-fold during early stationary phase at 28 MPa but downregulated 1.7-fold in log phase . To harmonize data, researchers should standardize culture conditions: OD = 1.5, 17°C, and 28 MPa for 72 hours . Conflicting solubility reports (e.g., 40% soluble vs. 15% in early studies) are attributable to buffer composition; Tris-HCl (pH 7.5) with 300 mM NaCl and 5% glycerol improves solubility by 55% .
How to design a robust experiment analyzing rplN-rRNA interactions?
Step 1: Perform electrophoretic mobility shift assays (EMSAs) with fluorescently labeled 23S rRNA fragments (nucleotides 1200–1350).
Step 2: Use isothermal titration calorimetry (ITC) to quantify binding affinity (K). Reported K values range from 12 nM (ambient pressure) to 28 nM (30 MPa) .
Step 3: Validate with in vitro reconstitution of 50S subunits, monitoring assembly kinetics via sucrose gradient centrifugation .
What statistical approaches are appropriate for pressure-response experiments?
For proteomic datasets (e.g., label-free LC-MS), apply arcsinh transformation to normalize intensity distributions before ANOVA . Pressure-dependent growth curves require nonlinear regression modeling (e.g., Gompertz equation) to estimate lag phases and maximal rates . When comparing structural models, use Ramachandran plot Z-scores with a threshold of −2.5 for outlier rejection .
Why do some studies report rplN as nonessential while others highlight critical roles?
Essentiality depends on environmental context. In nutrient-rich media at 0.1 MPa, ΔrplN mutants show only 20% growth reduction, masking its importance . Under simultaneous cold (4°C) and high-pressure (28 MPa) stress, viability drops to 5% of wild-type levels . Researchers must contextualize gene essentiality within experimental conditions.
How to reconcile discrepancies in post-translational modification (PTM) reports?
Phosphoproteomic studies identified conflicting phosphorylation at Ser32 (reported in but absent in ). This variability arises from phosphatase activity during lysis; adding 50 mM sodium fluoride to lysis buffers inhibits phosphatases, ensuring PTM preservation .
