Recombinant Escherichia coli O17:K52:H18 GTPase Era (era)

What is GTPase Era and what is its fundamental role in Escherichia coli?

GTPase Era (Essential Ras-like protein) is a deeply conserved protein that functions critically in the assembly of bacterial-type ribosomes, spanning from Escherichia coli to human mitochondria. It belongs to the TRAFAC (translation factors) class of GTPases and serves as a molecular switch rather than a GDP/GTP sensor as previously speculated . The protein is essential for bacterial viability, and its absence or dysfunction leads to severe physiological consequences. Era plays a pivotal role in the early stages of small ribosomal subunit (SSU) biogenesis, where it helps shape the platform region necessary for efficient translation . This function positions Era as a central nexus in protein synthesis regulation. Its activity is controlled through sophisticated mechanisms at transcriptional, post-transcriptional, and post-translational levels, highlighting its importance in cellular homeostasis . Furthermore, Era's function extends beyond simple ribosomal assembly to include broader roles in cellular physiology and stress adaptation.

How does the structure of GTPase Era relate to its function?

GTPase Era possesses a distinctive two-domain architecture consisting of an N-terminal GTPase domain and a C-terminal RNA-binding KH (K homology) domain connected by a flexible linker. The GTPase domain contains characteristic G-motifs (G1-G5) responsible for nucleotide binding and hydrolysis, while the KH domain specifically recognizes the 3′-minor domain of the small subunit ribosomal RNA . Era lacks an important Gln residue in the switch II region normally responsible for aligning water for nucleophilic attack in other GTPases, making it a "HAS-GTPase" (hydrophobic amino acid substituted) . This structural feature results in poor intrinsic GTPase activity, which is typically stimulated by potassium ions coordinated by invariant Asn residues in the G1 motif and the "K-loop" embedded in switch I . The conformational state of the GTPase domain, dependent on the bound nucleotide (GTP, GDP, or nucleotide-free), influences the orientation of the KH domain and consequently its RNA-binding capacity. X-ray crystallographic studies reveal that in the apo- or GDP-bound state, the KH domain is rotated such that a negatively charged helix partially blocks RNA access, while in the GTP-bound state, the domain reorients to allow unobstructed RNA binding . This conformational switching mechanism appears central to Era's function in ribosome assembly.

What are the recommended protocols for expressing and purifying recombinant GTPase Era from E. coli?

The expression and purification of recombinant GTPase Era typically begins with cloning the era gene into an appropriate expression vector containing an affinity tag (such as His6, GST, or MBP) to facilitate purification. The construct is then transformed into an expression strain of E. coli, with BL21(DE3) or its derivatives being common choices due to their reduced protease activity. Expression conditions require careful optimization, with typical induction using 0.1-1.0 mM IPTG at mid-log phase (OD600 ~0.6-0.8) followed by growth at lower temperatures (16-25°C) to enhance proper folding . Purification generally involves multiple chromatographic steps, beginning with affinity chromatography based on the chosen tag, followed by ion exchange chromatography to separate differently charged species, and finally size exclusion chromatography to achieve high purity and remove aggregates. Special attention must be paid to buffer composition throughout the purification process, as Era activity is influenced by potassium ions . The purification buffer typically contains 20-50 mM Tris or HEPES (pH 7.5-8.0), 100-300 mM KCl or NaCl, 5-10% glycerol, and 1-5 mM β-mercaptoethanol or DTT to maintain reduced cysteines. Adding 5-10 mM MgCl2 is crucial to maintain proper folding of the GTPase domain, while including 50-100 μM GDP during purification can enhance stability . The final purified protein should be flash-frozen in liquid nitrogen and stored at -80°C in small aliquots to minimize freeze-thaw cycles.

How can researchers effectively assess the GTPase activity of Era in vitro?

The assessment of Era's GTPase activity in vitro can be conducted using several complementary approaches, each with specific advantages. The most common method employs a colorimetric assay based on malachite green, which detects inorganic phosphate released during GTP hydrolysis. This approach requires carefully optimized reaction conditions: 50 mM Tris-HCl (pH 7.5), 50-100 mM KCl (crucial as a cofactor), 5 mM MgCl2, 0.1-1 μM purified Era protein, and 50-200 μM GTP . Reactions are typically conducted at 30-37°C for varying time periods before being stopped with malachite green reagent for phosphate detection. Alternative methods include HPLC-based separation of GTP from GDP, which offers higher sensitivity but requires specialized equipment, or coupled-enzyme assays that link phosphate release to NADH oxidation for real-time monitoring via absorbance at 340 nm. Radioactive assays using [γ-32P]GTP provide exceptional sensitivity for measuring the low intrinsic activity of Era but require radioisotope handling capabilities . When examining factors that influence GTPase activity, researchers should systematically vary potassium ion concentration (0-200 mM), test different divalent cations (Mg2+, Mn2+, Ca2+), and investigate potential regulatory interactions with 16S rRNA fragments or other binding partners. Temperature dependence should be assessed between 15-45°C, and pH optimum determined across the range of 6.0-9.0. Control experiments should include a catalytically inactive Era variant (such as K21R in E. coli Era) to establish background phosphate release rates .

What experimental approaches can be used to study Era-rRNA interactions?

Studying Era-rRNA interactions requires a multi-faceted experimental approach to capture both qualitative binding characteristics and quantitative binding parameters. Electrophoretic mobility shift assays (EMSA) provide a straightforward method to visualize complex formation between purified Era and in vitro transcribed 16S rRNA fragments, typically focusing on the conserved 3'-terminal region containing the CCUCC sequence recognized by Era's KH domain . Filter-binding assays offer a more quantitative alternative, allowing determination of binding constants when performed with radioactively labeled RNA. For higher-resolution binding data, surface plasmon resonance (SPR) or microscale thermophoresis (MST) can measure binding kinetics and affinities in real-time without labeling requirements. Structural insights into the interaction can be obtained through X-ray crystallography of Era-rRNA complexes, though this requires successful co-crystallization . Cryo-electron microscopy has emerged as a powerful technique to visualize Era bound to nascent ribosomal subunits in different assembly states. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map interaction interfaces by identifying regions of Era that become protected from solvent upon RNA binding. Researchers should systematically examine how the nucleotide-bound state (apo, GDP, GTP, or non-hydrolyzable GTP analogs) affects the RNA-binding properties of Era, as contradictory observations exist regarding whether nucleotide binding enhances or inhibits RNA binding . Mutational analysis targeting key residues in the KH domain should be performed to establish the specificity determinants of the interaction. Controls should include non-specific RNA sequences and careful buffer optimization, as ionic strength significantly influences electrostatic RNA-protein interactions.

How can researchers address apparent contradictions in Era-ribosome association data?

Addressing the apparent contradictions in Era-ribosome association data requires a systematic experimental approach combining biochemical, structural, and in vivo methodologies. The central contradiction involves observations that GTP binding enhances RNA binding capacity through KH domain reorientation, yet in vitro studies show apo-Era binds 16S rRNA more effectively than nucleotide-bound forms . To resolve this, researchers should first establish standardized biochemical conditions for binding studies, carefully controlling ionic strength, pH, temperature, and magnesium concentrations, as these significantly impact both protein-RNA and protein-nucleotide interactions. Time-resolved experiments that monitor the assembly process sequentially could reveal whether Era association with rRNA precedes nucleotide binding in a defined order. Single-molecule fluorescence resonance energy transfer (smFRET) experiments can track conformational changes in real-time as Era interacts with both nucleotides and rRNA targets . Hydrogen-deuterium exchange mass spectrometry (HDX-MS) would identify which Era regions become protected in different nucleotide-bound states with and without rRNA. Cryo-electron microscopy of ribosome assembly intermediates at different maturation stages could reveal the sequential binding and conformational changes of Era during the assembly process . Researchers should develop in vivo assays using fluorescently tagged Era variants to monitor their dynamic association with nascent ribosomes in living cells. Mutational analysis targeting the interface between the GTPase and KH domains could define how conformational information is transmitted between these regions. To determine if Era behavior differs between bacterial and mitochondrial systems, parallel studies with bacterial Era and mammalian ERAL1 should be conducted under identical conditions . Reconciling these contradictions will likely require recognizing that Era's function involves a more complex cycle of conformational states than the binary switch model initially suggested.

What mutational strategies can reveal the functional importance of specific GTPase Era residues?

Mutational analysis represents a powerful approach to dissect the structure-function relationships within GTPase Era. Researchers should begin with site-directed mutagenesis targeting conserved residues in key functional domains, with mutations designed to test specific mechanistic hypotheses. Within the GTPase domain, mutations in the G1 motif (P-loop) such as K21R in E. coli Era can disrupt GTP binding without affecting protein folding, while mutations in the G3 motif (Switch II) can impair GTP hydrolysis while preserving binding . Mutations in the switch regions can be designed to either mimic the GTP-bound state (activating) or the GDP-bound state (inactivating). Within the KH domain, mutations targeting residues that directly contact the 16S rRNA, particularly those interacting with the conserved CCUCC sequence, can reveal specificity determinants for RNA recognition . The interface between the GTPase and KH domains contains residues critical for transmitting conformational changes, making them valuable targets for mutagenesis to understand the allosteric mechanism. Beyond single point mutations, researchers should consider creating chimeric proteins, swapping domains between Era homologs from different species to identify species-specific functional elements. Double mutant cycles can reveal functional interactions between residues that might not be apparent from crystal structures . To rigorously assess mutational effects, a comprehensive phenotypic analysis should include: growth curves under various conditions (temperature, carbon sources), microscopic examination for morphological defects, ribosome profiles to assess subunit assembly, and in vitro assays for GTPase activity and RNA binding. Viability tests under depletion conditions can determine whether mutations create conditional or null phenotypes. Based on previous studies, even apparently conservative mutations in critical motifs (like K21R) can produce lethal phenotypes, highlighting the exquisite sensitivity of Era function to structural perturbations .

What are the key considerations for studying the role of Era in stress adaptation?

Studying Era's role in stress adaptation requires examining how this GTPase functions as a regulatory node linking ribosome assembly to cellular stress responses. Researchers should begin by characterizing Era expression and activity across diverse stress conditions (heat shock, cold shock, oxidative stress, nutrient limitation, stationary phase) using techniques like qRT-PCR for transcript levels and western blotting with phospho-specific antibodies to detect potential post-translational modifications . The relationship between Era and the stringent response deserves particular attention, as the alarmone (p)ppGpp may directly interact with Era to coordinate ribosome assembly with nutrient availability. In vitro binding assays can determine if (p)ppGpp binds Era and how this affects its GTPase activity and RNA-binding properties. Constructing conditional era mutants or utilizing controlled depletion systems permits examination of how Era deficiency impacts stress survival without the confounding effects of lethality. Temperature-sensitive mutants are particularly valuable for studying cold sensitivity, which is a common phenotype of era mutations . Ribosome profiling experiments comparing wild-type and era mutant strains under stress conditions can reveal how Era influences the translational program during adaptation. Genetic interaction screens using synthetic genetic arrays (SGA) can identify genes that become essential in era mutant backgrounds during stress, revealing functional relationships within stress response networks. Metabolomic profiling would determine if Era dysfunction alters stress-related metabolite levels. Since Era mutations often cause filamentation, microscopic techniques including fluorescence microscopy with cell division markers can connect Era function to morphological stress responses . As Era interacts with the SSU of ribosomes, researchers should examine whether stress-induced changes in 16S rRNA modification patterns affect Era binding and function using techniques like primer extension analysis or RNA-seq.

How should researchers analyze and interpret contradictory data about Era's nucleotide-dependent conformational states?

Analyzing contradictory data regarding Era's nucleotide-dependent conformational states requires a systematic approach that integrates multiple experimental techniques with careful consideration of experimental conditions. Researchers should begin by cataloging all available structural data (X-ray, cryo-EM, NMR) alongside biochemical findings, noting specific experimental conditions for each study, particularly buffer composition, protein concentration, and the presence of stabilizing agents that might influence conformational equilibria . Molecular dynamics simulations can bridge static structural snapshots to reveal accessible conformational space and energy barriers between different states. Nuclear magnetic resonance (NMR) spectroscopy is particularly valuable for analyzing protein dynamics in solution, potentially revealing populations of conformational states missed in crystallographic studies . Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map conformational flexibility across different nucleotide-bound states in solution. Time-resolved experiments using stopped-flow techniques coupled with fluorescence or FRET can track conformational transitions in real-time after nucleotide addition or displacement. When analyzing binding data, researchers should consider the possibility of negative cooperativity between nucleotide and RNA binding, which could explain seemingly contradictory observations . The apparent incongruity between bacterial and mitochondrial Era behavior should prompt careful comparison studies using identical experimental conditions. Mathematical modeling of Era's conformational cycle, incorporating all available kinetic and thermodynamic parameters, can generate testable hypotheses about transition states not directly observed experimentally. Researchers should systematically investigate how experimental conditions (ionic strength, temperature, divalent cation concentration) affect the observed conformational preferences, as these could explain discrepancies between studies . The final interpretation should acknowledge that the binary "switch" model might be oversimplified, with Era potentially cycling through multiple functionally relevant conformational states during ribosome assembly.

What statistical approaches are most appropriate for analyzing Era GTPase activity data?

The analysis of Era GTPase activity data requires robust statistical approaches tailored to the characteristics of enzyme kinetic measurements. Researchers should begin by establishing whether the data follow Michaelis-Menten kinetics, plotting initial velocities against substrate concentration and using non-linear regression to determine kinetic parameters (Km, Vmax, kcat) . When comparing wild-type Era to mutant variants or examining the effects of potential regulators, analysis of variance (ANOVA) followed by appropriate post-hoc tests (Tukey's HSD or Dunnett's test) should be employed to identify statistically significant differences. For time-course experiments measuring GTP hydrolysis, researchers should fit data to appropriate kinetic models (single-phase, two-phase, or more complex models) and compare goodness-of-fit using Akaike Information Criterion (AIC) or similar metrics . When analyzing the effects of varying conditions (pH, temperature, ion concentration) on Era activity, response surface methodology (RSM) can identify optimal conditions and interaction effects. Due to Era's low intrinsic GTPase activity, signal-to-noise ratios can be challenging; researchers should perform power analysis to determine the necessary number of replicates for detecting physiologically relevant differences . Regression analysis can quantify relationships between GTPase activity and various factors like potassium concentration, which stimulates Era activity approximately 10-fold . For complex datasets involving multiple variables, multivariate statistical approaches such as principal component analysis (PCA) or partial least squares regression (PLS) can reveal underlying patterns and correlations. Time-series analysis techniques are valuable for experiments tracking GTPase activity over extended periods. When comparing GTPase activities across different experimental techniques (colorimetric, HPLC, radioactive), researchers should establish correction factors through calibration curves using common standards. Outlier detection and robust statistical methods should be employed to handle the inherent variability in enzymatic assays, and all analyses should include appropriate control experiments to establish baseline activity levels.

What are the key considerations for interpreting phenotypic data from Era mutants?

Interpreting phenotypic data from Era mutants requires careful consideration of the multifaceted roles of this essential GTPase in cellular physiology. Researchers should first establish whether phenotypes result directly from altered Era function or from secondary effects, using complementation studies with wild-type era expressed from plasmids to confirm causality . The pleiotropic nature of Era dysfunction means that observed phenotypes could stem from different aspects of its function; researchers should therefore characterize multiple phenotypic parameters: growth rates in different conditions, cell morphology, ribosome profiles, stress resistance, and macromolecular synthesis rates. Temperature-dependent phenotypes are particularly informative, as many Era mutations cause cold sensitivity due to defects in ribosome assembly at lower temperatures . The severity of phenotypes should be correlated with biochemical measurements of GTPase activity and RNA binding to establish structure-function relationships. When interpreting growth defects, researchers should distinguish between effects on lag phase, exponential growth rate, and final cell density, as these reflect different aspects of cellular physiology. For morphological phenotypes like filamentation, quantitative image analysis should be employed to measure cell length distributions rather than subjective assessments . Transcriptomic and proteomic analyses can reveal compensatory responses to Era dysfunction, providing insights into cellular homeostatic mechanisms. Since Era affects ribosome assembly, translational responses measured by ribosome profiling may differ from transcriptional responses, making integrated multi-omics approaches particularly valuable. Synthetic genetic interaction screens can place Era within broader cellular networks by identifying genes whose deletion exacerbates or suppresses era mutant phenotypes . When interpreting phenotypic data, researchers should consider the possibility of gain-of-function effects in addition to loss-of-function, particularly for mutations that may lock Era in specific conformational states. The evolutionary conservation of Era means that comparative studies across species can help distinguish universal functions from species-specific roles.

What emerging technologies hold promise for advancing our understanding of GTPase Era function?

Several cutting-edge technologies are poised to transform our understanding of GTPase Era function in the coming years. Cryo-electron tomography (cryo-ET) combined with subtomogram averaging offers unprecedented potential to visualize Era's interactions with nascent ribosomes in their native cellular context, potentially revealing assembly intermediates previously inaccessible to conventional structural methods . Single-molecule techniques, including fluorescence resonance energy transfer (smFRET) and high-speed atomic force microscopy (HS-AFM), can track Era's conformational dynamics in real-time, potentially capturing transient states in the GTPase cycle that have eluded traditional structural approaches. AlphaFold2 and other AI-based protein structure prediction tools may help model Era complexes with interacting partners where experimental structures remain elusive . Time-resolved X-ray crystallography and X-ray free-electron laser (XFEL) techniques could capture Era in multiple conformational states during its functional cycle. Proximity labeling approaches (BioID, APEX) implemented in vivo can map Era's dynamic interactome across different growth conditions, potentially identifying unknown regulatory partners. CRISPR interference (CRISPRi) systems allowing titratable gene repression provide new tools for studying essential genes like era without the complications of traditional knockout approaches . Advanced mass spectrometry methods, such as cross-linking mass spectrometry (XL-MS) and native mass spectrometry, can capture Era in complex with nucleotides and rRNA under near-physiological conditions. Microfluidic techniques coupled with time-lapse microscopy enable tracking of single-cell responses to Era depletion, revealing cell-to-cell variability masked in population studies. Ribosome profiling during Era depletion could reveal specific translational effects that connect ribosome assembly defects to broader cellular phenotypes . Combinatorial genetics through CRISPR-based screens may uncover synthetic interactions that place Era within broader cellular networks controlling growth and stress adaptation.

What theoretical models might reconcile contradictory observations about Era's nucleotide-dependent ribosome binding?

Reconciling contradictory observations about Era's nucleotide-dependent ribosome binding requires developing theoretical models that extend beyond the simple binary switch paradigm. A promising "sequential binding model" posits that Era initially associates with nascent ribosomes in the apo-state, followed by GTP binding that induces conformational changes required for subsequent steps in ribosome maturation . This model aligns with observations that apo-Era binds 16S rRNA more effectively than nucleotide-bound forms in vitro, while also accommodating structural data showing GTP-dependent KH domain reorientation. An alternative "assembly-dependent conformational selection model" suggests that the ribosome assembly state itself influences Era's nucleotide preference, with early assembly intermediates favoring apo-Era binding while later intermediates require the GTP-bound form . This could explain why Era appears in different nucleotide states across various studies examining different ribosomal assembly stages. A "cooperative allostery model" proposes that rRNA binding and nucleotide binding exhibit complex cooperative or anti-cooperative relationships, with initial rRNA binding potentially altering Era's nucleotide preferences through allosteric mechanisms . This model could explain why Era's behavior differs in isolation versus in ribosomal contexts. A "multi-step conformational transition model" suggests Era cycles through multiple distinct conformational states beyond the canonical GDP/GTP dichotomy, with some conformations having no bound nucleotide yet resembling the "active" state structurally . This aligns with recent observations of nucleotide-free ERAL1 in mitochondrial SSU assembly intermediates. A comprehensive "integrated checkpoint model" proposes that Era functions as a quality control factor, with its GTPase activity triggered only when correct ribosome assembly has occurred, explaining why different experimental setups yield varying results based on whether they recapitulate the complete assembly environment . These theoretical frameworks generate testable predictions that could guide future experiments using time-resolved techniques to capture Era's dynamic cycling between states during ribosome assembly.

How might the study of GTPase Era inform broader understanding of ribosome assembly regulation?

The study of GTPase Era offers a valuable window into the broader mechanisms regulating ribosome assembly across all domains of life. As one of several GTPases involved in ribosome assembly, Era represents a model for understanding how energetic inputs (GTP hydrolysis) drive conformational changes that advance assembly from one state to the next, effectively creating irreversible steps in an otherwise reversible process . The remarkable conservation of Era from bacteria to human mitochondria suggests fundamental principles of assembly regulation that have withstood billions of years of evolutionary divergence. Comparative studies between bacterial Era and its mitochondrial homolog ERAL1 can reveal how ribosome assembly checkpoints have been preserved or modified as endosymbiotic bacteria evolved into organelles . The documented connection between Era dysfunction and the Perrault syndrome in humans (involving sensorineural deafness and ovarian dysgenesis) illuminates how ribosome assembly defects propagate to tissue-specific pathologies, informing our understanding of ribosomopathies more broadly . The integration of Era activity with the stringent response in bacteria through potential (p)ppGpp interaction exemplifies how ribosome assembly is coordinated with broader cellular physiology and stress responses. Understanding how Era monitors the correct formation of the 3' minor domain of 16S rRNA provides insights into the hierarchical nature of quality control during assembly, with specific factors checking distinct structural features before allowing progression . The potentially ordered recruitment and release of multiple assembly factors, including Era, suggests the existence of an "assembly code" that ensures correct sequential maturation of ribosomal subunits. The involvement of similar GTPases in both prokaryotic and eukaryotic ribosome assembly points to universal principles of energy-dependent assembly checkpoints across all cellular life . The study of Era's precise binding site near the decoding center of the small subunit reveals how assembly factors can influence the functional centers of the mature ribosome.

What are the implications of Era research for developing novel antimicrobial strategies?

The essential nature of GTPase Era in bacterial viability positions it as a promising target for novel antimicrobial development, with several strategic advantages over traditional antibiotic targets. Era's high conservation across bacterial species suggests that inhibitors could potentially have broad-spectrum activity, while its absence in the cytoplasm of eukaryotic cells (though present in mitochondria) provides a basis for selective toxicity . Structure-based drug design approaches can exploit the unique features of Era's GTPase domain, particularly the absence of the canonical Gln residue in switch II that makes it a HAS-GTPase, potentially allowing for selective targeting distinct from human GTPases . The nucleotide-binding pocket offers a well-defined target for competitive inhibitors, while the interface between the GTPase and KH domains presents opportunities for allosteric inhibitors that could lock Era in inactive conformations. High-throughput screening campaigns using GTPase activity assays can identify lead compounds from diverse chemical libraries. Fragment-based drug discovery approaches are particularly suited for GTPase targets, allowing the building of inhibitors with optimal properties . The involvement of Era in ribosome assembly suggests that inhibitors would disrupt multiple cellular processes simultaneously, potentially reducing the emergence of resistance. In vitro ribosome assembly assays incorporating Era can serve as secondary screens to validate the on-target effects of potential inhibitors. Conditional era mutants in model bacteria provide valuable tools for validating target engagement in vivo, potentially through synthetic lethality approaches . Since Era mutations often cause cold sensitivity, inhibitors might show enhanced efficacy at lower temperatures, suggesting potential applications for treating infections in the extremities or skin. For development purposes, careful structural comparison between bacterial Era and human ERAL1 is essential to design inhibitors that avoid mitochondrial toxicity. The association of ERAL1 dysfunction with Perrault syndrome highlights the importance of this selectivity consideration . Combination therapies targeting Era alongside other ribosome assembly factors or traditional antibiotics could provide synergistic effects and reduce resistance development.

How can researchers effectively troubleshoot problems in Era expression and purification?

Troubleshooting problems in the expression and purification of recombinant GTPase Era requires a systematic approach addressing common challenges specific to this protein. For poor expression yields, researchers should optimize codon usage for the expression host, as the era gene may contain rare codons that limit translation efficiency. Lowering induction temperature (16-20°C) and IPTG concentration (0.1-0.5 mM) can enhance proper folding by slowing protein production . Testing multiple expression strains, including those with extra copies of rare tRNAs (Rosetta) or enhanced disulfide bond formation (Origami), may improve yields. If Era forms inclusion bodies, solubility can be improved by co-expression with molecular chaperones (GroEL/ES, DnaK/J) or fusion to solubility-enhancing tags like MBP or SUMO. For purification difficulties, researchers should ensure buffers contain sufficient magnesium (5-10 mM MgCl2) to stabilize the GTPase domain . Including GDP (50-100 μM) throughout purification can enhance stability by maintaining a uniform conformational state. If the protein shows evidence of proteolytic degradation, additional protease inhibitors should be included in all buffers, and handling time should be minimized. For Era proteins showing low activity, researchers should verify nucleotide content, as tightly bound GDP from the expression host may need to be removed by EDTA treatment followed by buffer exchange before activity assays . If affinity tags adversely affect function, on-column tag cleavage protocols can be implemented to avoid additional handling steps. Size exclusion chromatography profiles showing unexpected oligomerization or aggregation may be addressed by optimizing buffer conditions, particularly ionic strength and reducing agent concentration. For long-term storage issues, glycerol concentration should be increased to 15-20%, and stability of different nucleotide-bound states (apo, GDP, GTP) should be systematically compared . If the purified protein shows poor solubility at concentrations needed for structural studies, screening buffer additives like arginine (50-100 mM) or mild detergents may improve behavior.

What experimental design approaches can address the regulatory status of research with recombinant E. coli expressing GTPase Era?

Designing experiments with recombinant E. coli expressing GTPase Era requires careful consideration of regulatory requirements and exemption criteria. Researchers should first determine whether their work falls under the exemptions in Section III-F of the NIH Guidelines, particularly examining whether the recombinant nucleic acids consist entirely of DNA segments from a single prokaryotic source . This assessment should be documented in laboratory records to demonstrate due diligence in regulatory compliance. The choice of host strain is critical; researchers should preferentially use well-characterized laboratory strains like E. coli K-12 derivatives that have established safety records and are specifically mentioned in regulatory guidelines . Expression vectors should be non-mobilizable and lack transfer mechanisms to prevent horizontal gene spread, preferably using vectors with comprehensive safety documentation. The experimental design should incorporate appropriate biological containment measures based on a risk assessment covering both the host organism and the expressed Era protein . Standard microbiological practices including proper waste decontamination, use of biohazard containers, and prevention of aerosol formation are essential regardless of exemption status. For large-scale cultivations exceeding 10 liters, researchers should consult institutional biosafety committees early in the planning process, as additional requirements may apply . The experimental protocol should include validation steps to confirm that the recombinant Era does not confer unexpected phenotypes that might alter the risk profile, such as enhanced survival under stress conditions. Detailed documentation should be maintained throughout the research, including strain construction, sequence verification, and containment procedures . When publishing research involving recombinant Era, materials and methods sections should include statements about regulatory compliance and risk assessment outcomes. For collaborative projects, especially international collaborations, researchers should account for potential differences in regulatory frameworks between jurisdictions. Prior consultation with institutional biosafety officers can help researchers navigate complex regulatory landscapes and identify any institutional requirements beyond national guidelines .