Recombinant Escherichia coli O9:H4 GTPase Era (era)

What is E. coli GTPase Era and what are its key structural domains?

Era (Escherichia coli Ras-like protein) is an essential GTP binding protein required for bacterial proliferation. Structurally, Era consists of two major functional domains: a typical GTPase/GTP-binding domain and a putative K homology (KH) domain that serves as an RNA binding region . This dual-domain architecture enables Era to coordinate GTP hydrolysis with RNA interaction, providing a potential regulatory link between energy status and cellular processes. Homologous sequences exist across diverse organisms including humans, mice, Drosophila, C. elegans, and plants, suggesting evolutionary conservation of this important protein family .

How does Era function in E. coli cellular processes?

Era plays critical roles in ribosome assembly, cell cycle regulation, and potentially coordinates these processes with cellular energy status through its GTPase activity. The KH domain enables specific RNA interactions that are essential for Era's biological function. Experimental evidence shows that mutations in the GTPase domain can dramatically affect cellular function, indicating the importance of GTP hydrolysis for proper Era activity . The C-terminal region containing part of the KH domain is particularly important, as studies with human ERA homologues demonstrate that deletion of this region alters functional outcomes .

What expression systems are most suitable for recombinant Era production?

For laboratory-scale production of recombinant Era, E. coli-based expression systems offer several advantages due to their rapid growth and ease of genetic manipulation. Commonly used E. coli strains include BL21(DE3) and its derivatives, which lack certain proteases that might degrade recombinant proteins. Expression vectors containing inducible promoters (T7, tac) with affinity tags (His, GST) facilitate controlled expression and subsequent purification. When designing expression systems, consideration should be given to:

How can factorial design methodology improve recombinant Era expression?

Traditional one-variable-at-a-time approaches to protein expression often fail to identify optimal conditions and require numerous experiments . Statistical factorial design offers a more efficient alternative by allowing simultaneous evaluation of multiple variables and their interactions . For Era expression, key parameters to include in factorial designs are:

• Temperature (typically testing 16°C, 25°C, and 37°C)

• Inducer concentration

• Media composition

• Induction timing

• Expression duration

This multivariant approach enables thorough analysis compared to traditional univariant methods, allowing estimation of statistically significant variables while accounting for interactions between them . For instance, a two-level factorial design with four factors requires only 16 experiments yet provides comprehensive data on main effects and interactions. The advantage of this approach is that many variables can be screened simultaneously with relatively few experimental trials, making it particularly valuable for optimizing recombinant protein expression .

What purification strategies yield functional recombinant Era protein?

Purification of active Era requires careful consideration of its biochemical properties:

The addition of nucleotides during purification often improves stability of GTPases like Era. For structural studies, additional considerations include buffer optimization through thermal shift assays and removal of affinity tags if they interfere with function or crystallization.

How can site-directed mutagenesis reveal structure-function relationships in Era?

Site-directed mutagenesis provides valuable insights into Era's molecular mechanisms. Based on homology studies, several key residues can be targeted:

• Conserved residues in the GTPase domain (G1-G5 motifs) to create GTPase-deficient mutants

• Key residues in the KH domain that mediate RNA binding

• Interface residues between domains to understand interdomain communication

Studies with human ERA homologues demonstrate that amino acid substitutions in the GTPase domain can induce apoptosis in mammalian cells, highlighting the importance of proper GTPase function . Additionally, deletion of the C-terminal region containing part of the KH domain alters functional outcomes, suggesting this domain is critical for ERA activity .

What methods best characterize Era's interaction with RNA substrates?

Era's KH domain mediates specific RNA interactions that are essential for its function. Experimental evidence confirms RNA binding activity through pull-down experiments using RNA homopolymer immobilized on beads and recombinant ERA proteins . For comprehensive characterization:

How can adaptive evolution approaches enhance Era expression systems?

Adaptive evolution offers a powerful strategy for improving recombinant protein production. Drawing from approaches used with recoded E. coli strains , researchers can:

• Culture Era-expressing strains for extended periods (>1,000 generations) in selective conditions

• Isolate colonies with improved growth characteristics

• Sequence genomes to identify beneficial mutations

• Reconstruct individual alleles via multiplex automatable genome engineering (MAGE) to quantify fitness effects

This approach has proven effective for improving strains with modified genetic codes, where evolved populations significantly exceeded growth rates of ancestral strains . For Era expression, adaptive evolution could select for mutations that improve tolerance to Era overexpression or enhance its folding and stability, ultimately leading to higher yields of functional protein.

What genomic analysis approaches reveal Era function across bacterial species?

Comparative genomics provides valuable insights into Era's evolutionary conservation and functional diversification:

• Multiple sequence alignments to identify conserved motifs and species-specific variations

• Phylogenetic analysis to trace evolutionary relationships

• Genomic context analysis to identify co-occurring genes and potential functional relationships

• Structural prediction to compare three-dimensional architectures across species

Recent genomic studies of E. coli demonstrate remarkable genomic plasticity with frequent gene acquisition and loss events . Similar approaches can reveal how Era has evolved across bacterial lineages and potentially identify species-specific adaptations in its function.

What strategies address inclusion body formation in recombinant Era expression?

Inclusion body formation is a common challenge when expressing recombinant proteins including Era. Several approaches can mitigate this issue:

The multivariant statistical approach described in search result is particularly valuable for systematic optimization, as it allows evaluation of multiple parameters simultaneously rather than changing one variable at a time, which often fails to identify optimal conditions .

How can researchers address inconsistent GTPase activity measurements?

Variability in Era GTPase activity assays can stem from multiple sources:

• Nucleotide content: Purified GTPases often contain a mixture of bound nucleotides (GDP/GTP)

  • Solution: Include nucleotide exchange steps before activity measurements

• Protein quality variation: Batch-to-batch differences in purity or folding

  • Solution: Implement rigorous quality control via SEC-MALS and circular dichroism

• Assay conditions: Buffer components significantly affect GTPase activity

  • Solution: Standardize assay conditions including Mg²⁺ concentration, pH, and temperature

• Detection method limitations: Different GTPase assay methods have varying sensitivities

  • Solution: Validate results using orthogonal detection methods (radioactive, colorimetric, HPLC)

How might Era research inform antibiotic development strategies?

As an essential bacterial protein with no direct human homolog, Era represents a potential antibiotic target. Future research directions could include:

• High-throughput screening for Era inhibitors

• Structure-based drug design targeting the GTPase active site

• Exploration of Era's role in bacterial persistence and antibiotic tolerance

• Investigation of Era as a target for combination therapy approaches

Studies examining genomic plasticity in clinical E. coli isolates have revealed frequent acquisition of resistance genes , highlighting the need for novel antibiotic targets like Era that are essential and highly conserved.

What emerging technologies will advance Era structure-function studies?

Several cutting-edge technologies promise to deepen our understanding of Era:

• Cryo-electron microscopy: For visualizing Era-ribosome complexes at near-atomic resolution

• Single-molecule approaches: To observe GTPase cycling and conformational changes in real-time

• In-cell NMR: For studying Era structure and interactions in the native cellular environment

• Advanced genetic approaches: CRISPR-based methods for precise genome manipulation

• Systems biology integration: Placing Era function within genome-scale metabolic models

How can synthetic biology approaches leverage Era for novel applications?

Era's essential role in bacterial growth regulation makes it an interesting target for synthetic biology applications:

• Engineered Era variants with altered GTPase activity as growth-rate modulators

• Synthetic circuits incorporating Era to achieve growth-dependent gene expression

• Era-based biosensors for monitoring cellular energy status

• Exploitation of Era's RNA-binding properties for synthetic RNA regulatory systems

These approaches could build upon recoded genome work that demonstrates the remarkable adaptability of E. coli to genetic modifications .