Recombinant Sulfolobus solfataricus 30S ribosomal protein S13P (rps13p)

What is Sulfolobus solfataricus 30S ribosomal protein S13P (rps13p)?

Sulfolobus solfataricus 30S ribosomal protein S13P (rps13p) is a component of the small ribosomal subunit in this hyperthermophilic archaeon. This protein contributes to ribosome assembly and function in translation processes at high temperatures, as S. solfataricus grows optimally at 80°C . Like other archaeal ribosomal proteins, it shares evolutionary characteristics with both bacterial and eukaryotic homologs, making it valuable for studying translation machinery evolution. When studying rps13p, it's essential to consider its native hyperthermophilic environment and the structural adaptations that enable its function under extreme conditions.

What expression systems are available for producing recombinant rps13p?

Two primary expression systems are suitable for producing recombinant proteins from S. solfataricus, including rps13p:

• Heterologous expression in E. coli: Typically using ROSETTA(DE3)/pLysS strains with vectors like pETM11 for N-terminal His-tagging . This system generally provides higher protein yields but may present challenges with proper protein folding.

• Homologous expression directly in S. solfataricus: Using the virus-based shuttle vector system developed for hyperthermophilic archaea, with vectors like pMJ05 for C-terminal His-tagging . This approach was specifically refined for high-level gene expression in S. solfataricus with two different promoters: the heat-inducible promoter of the major chaperonin (thermophilic factor 55) and the arabinose-inducible promoter of the arabinose-binding protein AraS .

The choice of system depends on research requirements - whether native folding or higher yield is the priority.

How does the hyperthermophilic nature of S. solfataricus affect rps13p purification?

The hyperthermophilic nature of S. solfataricus affects rps13p purification in several advantageous ways:

• Heat treatment can serve as an initial purification step. When expressed in E. coli, heating the lysate to 65-75°C denatures most host proteins while leaving thermostable rps13p intact.

• Buffer compositions must accommodate thermostability, typically requiring higher salt concentrations (100-500 mM NaCl) and stabilizing agents.

• Purification procedures can be conducted at elevated temperatures, which may improve efficiency by reducing contaminating protein binding to chromatography matrices.

• The inherent stability of rps13p at high temperatures results in longer shelf-life of the purified protein compared to mesophilic counterparts.

When purifying rps13p from S. solfataricus directly, specialized lysis buffers containing Tris-HCl pH 7.4 50 mM, NaCl 100 mM, PMSF 1 mM, DTT 1 mM, MgAcetate 10 mM, and Triton X-100 0.1% have proven effective .

What tagging strategies work best for rps13p purification?

Based on established protocols for S. solfataricus proteins, the optimal tagging strategies for rps13p include:

The research demonstrates successful single-step purification with His-tagged proteins using affinity chromatography . For ribosomal proteins like rps13p, tag placement should consider potential interference with ribosome incorporation or RNA binding. Additionally, incorporating a protease cleavage site between the tag and protein enables tag removal for studies requiring the native protein structure.

How can I optimize cell growth and lysis for S. solfataricus expressing rps13p?

For optimal growth and lysis of S. solfataricus expressing rps13p, follow this methodological approach:

• Culture conditions:

  • Grow S. solfataricus at 75°C in Brock's medium supplemented with 0.2% NZ amine and 0.2% sucrose

  • Monitor growth until culture reaches OD600 of 0.6-0.8

  • For inducible systems, add appropriate inducer (heat-shock or arabinose)

• Cell harvesting:

  • Pellet cells by centrifugation

  • Resuspend in lysis buffer: Tris-HCl pH 7.4 50 mM; NaCl 100 mM; PMSF 1 mM; DTT 1 mM; MgAcetate 10 mM; Triton X-100 0.1%

• Cell lysis:

  • Apply sonication for effective disruption of S. solfataricus cells

  • Clarify extract by centrifugation at 26,000× g for 30 min at 4°C

  • Collect supernatant and subject to ultracentrifugation at 100,000× g for 1 h at 4°C to obtain S100 extract

• Critical considerations:

  • Maintain sterile conditions throughout

  • Include protease inhibitors to prevent degradation

  • Consider oxygen-free conditions if the protein contains sensitive cysteine residues

  • Prepare fresh buffers to avoid contamination issues

These protocols have been successfully used for extracting proteins from S. solfataricus and can be adapted specifically for rps13p purification .

How can I study rps13p's interaction with RNA?

To study rps13p's interaction with RNA, implement these methodological approaches:

• Immunoprecipitation of native rps13p-RNA complexes:

  • Develop specific antibodies against rps13p

  • Incubate S. solfataricus S100 lysate with anti-rps13p antibodies

  • Use Protein G Dynabeads for pulldown

  • Extract bound RNAs using phenol/chloroform/isoamyl alcohol treatment

  • Analyze RNA by RT-PCR or sequencing

• In vitro binding studies:

  • Generate recombinant rps13p with appropriate tags

  • Prepare RNA substrates through in vitro transcription

  • Conduct binding assays at physiologically relevant temperatures (65-80°C)

  • Analyze complex formation through electrophoretic mobility shift assays

• Structural analysis of complexes:

  • Use SAXS to detect conformational changes upon RNA binding, similar to methods used for aIF5A

  • Apply computational modeling to predict binding interfaces

• Controls and validation:

  • Use pre-immune serum as a negative control for immunoprecipitation

  • Include RNase treatment controls to confirm specificity

  • Verify RNA recovery with established markers

This comprehensive approach can reveal both the identity of RNA partners and the nature of their interaction with rps13p.

What techniques are suitable for structural characterization of rps13p?

For structural characterization of rps13p from S. solfataricus, several complementary approaches are recommended:

• Solution structure analysis using Small-Angle X-ray Scattering (SAXS):

  • Prepare protein samples at multiple concentrations (0.5-10 mg/mL)

  • Test different buffer conditions (varying salt concentrations)

  • Collect data at temperatures approaching physiological conditions (up to 65°C)

  • Process data to obtain information about oligomeric state and conformational changes

• Computational modeling:

  • Use SwissModel or AlphaFold2 to build 3D structural models

  • Perform comparative modeling using templates from related archaeal proteins

  • Analyze electrostatic surface potential using PDB2PQR and APBS tools to identify RNA-binding interfaces

• Biophysical characterization:

  • Circular dichroism for secondary structure analysis

  • Thermal stability studies at varying temperatures

  • Limited proteolysis to identify domain boundaries

• High-resolution structural approaches:

  • X-ray crystallography screening

  • Cryo-EM studies, particularly valuable for visualizing rps13p within the assembled 30S ribosomal subunit

These methods can provide complementary information about rps13p's structure and functional interactions.

How can I investigate potential ribonuclease activity of rps13p?

To investigate potential ribonuclease activity of rps13p, follow these methodological steps:

• RNA degradation assays:

  • Pre-activate purified rps13p for 10 min at 65°C

  • Prepare different RNA substrates (rRNA, tRNA, mRNA)

  • Heat RNA samples briefly (5 min at 85°C) before adding to reaction

  • Incubate rps13p with RNA in buffer (10 mM HEPES pH 8.0, 100 mM KCl, 5 mM MgCl2, 5 mM β-mercaptoethanol, 5% glycerol)

  • Conduct reactions at 65°C for 30 min

  • Stop reactions by adding RNA loading dye

  • Analyze degradation patterns on agarose gels

• Substrate specificity determination:

  • Test structured RNAs (like 23S and 16S rRNA from S. solfataricus ribosomal subunits)

  • Compare with shorter, less structured RNAs (like tRNAs)

  • Examine sequence-specific effects using defined RNA transcripts

• Activity validation:

  • Perform zymogram assays with total rRNA included in gel matrix

  • Include appropriate controls (RNA without protein, heat-inactivated protein)

This approach parallels successful methods used to characterize ribonuclease activity in other S. solfataricus proteins like aIF5A .

How does rps13p structure and function compare across archaeal species?

To conduct comparative analysis of rps13p across archaeal species:

• Sequence analysis:

  • Perform multiple sequence alignments using tools like COBALT

  • Identify conserved domains and variable regions

  • Construct phylogenetic trees to visualize evolutionary relationships

• Structural comparison:

  • Generate 3D models for rps13p from different archaeal species

  • Superimpose structures to identify conserved structural elements

  • Analyze electrostatic surfaces to compare RNA binding potential

• Thermostability determinants:

  • Compare amino acid composition profiles (higher Glu/Lys content in thermophiles)

  • Identify unique structural features like additional salt bridges

  • Analyze hydrophobic core packing differences

• Functional complementation:

  • Express rps13p homologs in S. solfataricus

  • Test their ability to incorporate into functional ribosomes

  • Measure translation efficiency with different homologs

• Molecular dynamics simulations:

  • Analyze protein stability at different temperatures

  • Compare flexibility and rigidity of different homologs

This comparative approach can provide insights into both the universally conserved features of S13 proteins and the specific adaptations in hyperthermophiles.

What challenges arise when analyzing rps13p within the assembled ribosome?

Analysis of rps13p within the assembled ribosomal context presents several methodological challenges:

• Ribosome isolation considerations:

  • S. solfataricus ribosomes must be prepared carefully, following established protocols

  • During ultracentrifugation on 10-30% sucrose gradients, separation of 50S and 30S subunits can be achieved

  • Buffer conditions must maintain ribosome integrity at elevated temperatures

• Structural analysis limitations:

  • Cryo-EM studies must account for the unusual composition of archaeal ribosomes

  • Sample preparation may require stabilization strategies to prevent dissociation

  • Data processing needs to account for heterogeneity in ribosome populations

• Functional studies complexity:

  • In vitro translation systems for hyperthermophiles are technically challenging

  • Separating the contribution of individual proteins like rps13p requires sophisticated depletion and reconstitution approaches

  • Temperature-dependent effects complicate interpretation of results

• Protein-protein interaction network analysis:

  • Cross-linking studies must be optimized for thermophilic conditions

  • Multiple ribosomal proteins may have overlapping functions

  • Distinguishing direct and indirect effects requires careful controls

How can I resolve contradictory data about rps13p function?

When faced with contradictory data regarding rps13p function, implement these systematic approaches:

• Methodological standardization:

  • Document all experimental parameters in detail (temperatures, buffer compositions, protein concentrations)

  • Establish standard operating procedures for key experiments

  • Use consistent protein preparation methods

• Expression system comparison:

  • Compare results from E. coli-expressed vs. S. solfataricus-expressed rps13p

  • Evaluate the impact of different tags on protein function

  • Check for post-translational modifications that might differ between systems

• Condition-dependent effects:

  • Test function across temperature ranges (25-85°C)

  • Vary salt concentrations to identify ionic strength dependencies

  • Examine pH effects on activity

• Protein quality assessment:

  • Verify protein folding using circular dichroism

  • Check oligomeric state using SAXS or size exclusion chromatography

  • Assess RNA contamination that might affect functional assays

• Cross-validation strategies:

  • Apply multiple complementary techniques to test the same hypothesis

  • Collaborate with other laboratories for independent verification

  • Design experiments that can distinguish between competing models

This systematic troubleshooting approach can identify sources of variability and resolve seemingly contradictory results about rps13p function.

What approaches can reveal rps13p's role in hyperthermophilic translation?

To elucidate rps13p's specific role in hyperthermophilic translation, implement these advanced methodological approaches:

• Selective ribosome reconstitution:

  • Develop in vitro reconstitution systems for S. solfataricus 30S subunits

  • Compare translation activity with and without rps13p

  • Create rps13p variants with specific mutations to test functional hypotheses

• Temperature-dependent structural dynamics:

  • Use time-resolved techniques to capture conformational changes during translation

  • Compare dynamics at different temperatures (37°C vs. 75°C)

  • Identify temperature-sensitive steps in translation that may involve rps13p

• Translation factor interactions:

  • Examine rps13p interaction with initiation factors like aIF5A

  • Investigate how these interactions change with temperature

  • Develop fluorescence-based assays to monitor binding events in real-time

• In vivo approaches:

  • Develop conditional depletion systems for rps13p in S. solfataricus

  • Use ribosome profiling to identify translation changes upon rps13p depletion

  • Apply CRISPR-based approaches to introduce mutations at the genomic level

• Comparative translation systems:

  • Compare translation efficiency and accuracy between thermophilic and mesophilic systems

  • Swap rps13p between these systems to identify temperature-specific roles

  • Measure effects on specific steps of translation (initiation, elongation, termination)

These approaches can connect structural features of rps13p to specific functional roles in maintaining efficient translation under extreme temperature conditions.

Why might recombinant rps13p show inconsistent activity?

Inconsistent activity of recombinant rps13p can stem from several methodological issues:

• Expression-related factors:

  • Inclusion body formation in E. coli systems leading to improperly folded protein

  • Incomplete induction in S. solfataricus expression systems

  • Proteolytic degradation during expression or purification

• Purification considerations:

  • Inadequate heat treatment steps (temperature or duration)

  • Improper buffer composition affecting protein stability

  • Co-purification of inhibitory factors or RNA

  • Tag interference with functional domains

• Storage conditions:

  • Protein aggregation during freeze-thaw cycles

  • Oxidation of critical cysteine residues

  • Temperature-sensitive conformational changes

  • Buffer component degradation over time

• Activity assay variables:

  • Inconsistent reaction temperatures

  • Variation in substrate preparation

  • Batch-to-batch differences in reagents

  • Inadequate controls for non-specific activities

To address these issues, implement rigorous quality control measures at each step of protein production and characterization, and validate protein activity using multiple complementary assays.

What are optimal storage conditions for purified rps13p?

For maintaining optimal activity of purified rps13p, follow these evidence-based storage recommendations:

Unlike mesophilic proteins, thermostable proteins like rps13p often retain activity better after storage, but regular validation of activity remains essential. For working stocks that will undergo multiple freeze-thaw cycles, increasing glycerol content to 10-20% can provide additional protection.

How might rps13p research contribute to synthetic biology applications?

Research on rps13p from S. solfataricus has significant potential for synthetic biology applications in several areas:

• Thermostable translation systems:

  • Development of cell-free protein synthesis platforms operating at elevated temperatures

  • Creation of ribosomes with enhanced stability for industrial protein production

  • Engineering orthogonal translation systems with specialized functions

• Protein engineering applications:

  • Identification of thermostabilizing motifs that can be transferred to other proteins

  • Design of chimeric ribosomal proteins with novel functions

  • Creation of minimal functional ribosomes for synthetic cells

• Biotechnological tools:

  • Utilization of thermostable ribosomal components for diagnostic applications

  • Development of biosensors operating under extreme conditions

  • Creation of temperature-responsive regulatory systems

• Directed evolution platforms:

  • Using S. solfataricus expression systems for evolving proteins under extreme conditions

  • Developing selection methods based on translational efficiency at high temperatures

  • Creating libraries of rps13p variants with enhanced or modified functions

This research not only expands our understanding of archaeal translation but also provides valuable tools for the emerging field of extreme environment synthetic biology.

What are emerging technologies for studying thermophilic ribosomal proteins?

Several emerging technologies show promise for advancing research on thermophilic ribosomal proteins like rps13p:

• Advanced structural methods:

  • Time-resolved cryo-EM to capture translation intermediates

  • Single-particle FRET to observe conformational dynamics

  • Hydrogen-deuterium exchange mass spectrometry for identifying flexible regions

• In situ approaches:

  • Cryo-electron tomography of intact S. solfataricus cells

  • Super-resolution microscopy adapted for thermophilic organisms

  • In-cell NMR to observe protein behavior in native environment

• Genetic and genomic tools:

  • CRISPR-Cas systems adapted for archaeal genome engineering

  • Ribosome profiling at high temperatures

  • Selective ribosome profiling to identify rps13p-associated mRNAs

• Computational advances:

  • Molecular dynamics simulations at elevated temperatures

  • Machine learning approaches to predict thermal adaptation features

  • Systems biology models of archaeal translation

• Microfluidic applications:

  • Single-cell analysis of translation in thermophiles

  • Droplet-based assays for high-throughput functional testing

  • Gradient systems to assess temperature-dependent effects

These emerging technologies will enable researchers to address previously intractable questions about rps13p function within the context of hyperthermophilic translation.