Recombinant Callithrix jacchus Elongation factor 2 (EEF2), partial

What is Callithrix jacchus Elongation Factor 2 and what is its biological function?

Elongation Factor 2 (EEF2) in Callithrix jacchus (common marmoset) serves as a critical component in protein synthesis. It catalyzes the GTP-dependent ribosomal translocation step during translation elongation . This process is essential for protein synthesis across eukaryotes, facilitating the movement of the ribosome along the mRNA strand after peptide bond formation. The protein plays a fundamental role in maintaining proper translation rates and fidelity in marmoset cells.

Why are marmosets (Callithrix jacchus) valuable as research models?

The common marmoset (Callithrix jacchus) has gained prominence as an experimental model due to several advantageous characteristics. These small New World primates from Northeastern Brazil offer benefits including their compact size (300-500g), rapid reproduction rate, and specific brain features that make them suitable for various research applications . Their smaller size compared to traditional Old World primates provides practical advantages in laboratory settings while still offering relevant primate biology .

Marmosets have been successfully employed as models for numerous diseases and conditions, including:

• Infectious diseases: anthrax, tularemia, melioidosis, and viral infections (Lassa virus, eastern equine encephalitis virus)

• Neurological studies: retinal innervation and brain function research

• Hemorrhagic fever research: Marburg virus infection through various exposure routes

What are the key structural characteristics of partial recombinant EEF2?

Partial recombinant EEF2 refers to a fragment of the complete protein that contains specific functional domains rather than the entire protein sequence. While the search results don't provide specific structural information about Callithrix jacchus EEF2, this protein likely maintains the conserved GTP-binding domains and diphthamide modification site that are characteristic of EEF2 proteins across species. These structural elements are crucial for its ribosomal translocation function during protein synthesis.

What expression systems are optimal for producing recombinant Callithrix jacchus EEF2?

While the search results don't specifically address expression systems for Callithrix jacchus EEF2, they do mention that other recombinant proteins for marmoset research, such as myelin oligodendrocyte glycoprotein (MOG), are successfully expressed in Escherichia coli systems . For EEF2 expression, bacterial systems like E. coli would likely be suitable for producing the partial protein, particularly if post-translational modifications are not critical for the intended application.

The expression protocol would typically involve:

• Cloning the partial EEF2 sequence into an appropriate expression vector

• Transformation into a compatible E. coli strain

• Induction of protein expression under optimized conditions

• Cell lysis and initial purification steps

• Further purification through affinity chromatography or other suitable methods

For applications requiring post-translational modifications, mammalian or insect cell expression systems might be preferable, though these would involve more complex protocols and potentially lower yields.

How can researchers validate the biological activity of recombinant Callithrix jacchus EEF2?

Validation of recombinant EEF2 activity would require functional assays that assess its capability to perform GTP-dependent translocation. Key validation approaches include:

• GTP binding and hydrolysis assays to confirm the protein's ability to bind and hydrolyze GTP

• Ribosome binding assays to verify interaction with ribosomal components

• In vitro translation assays to demonstrate functional activity in promoting protein synthesis

• Structural analysis through circular dichroism or other techniques to confirm proper folding

These validation steps are essential before using the recombinant protein in more complex experimental systems to ensure that observed effects are attributable to functional EEF2 activity.

What experimental controls should be incorporated when studying recombinant Callithrix jacchus EEF2?

When designing experiments with recombinant Callithrix jacchus EEF2, appropriate controls should include:

• Negative controls:

  • Heat-inactivated EEF2 (to control for non-specific protein effects)

  • Buffer-only conditions

  • Non-functional EEF2 mutants (e.g., GTP-binding deficient variants)

• Positive controls:

  • Commercially available EEF2 from well-characterized sources

  • Native EEF2 purified from Callithrix jacchus tissues (when available)

  • EEF2 from closely related species with known activity

• Specificity controls:

  • Other elongation factors (e.g., EEF1) to demonstrate function specificity

  • Concentration gradients to establish dose-dependent effects

How can recombinant Callithrix jacchus EEF2 contribute to translational control studies?

Recombinant Callithrix jacchus EEF2 can serve as a valuable tool for investigating translational control mechanisms in marmoset models. Given that marmosets are used in neuroanatomical investigations and brain function studies , recombinant EEF2 could be particularly useful for examining translation regulation in neurological contexts.

Potential applications include:

• Investigating phosphorylation-dependent regulation of protein synthesis

• Examining species-specific aspects of translational control

• Studying the effects of neurodegenerative disease-associated mutations on translation efficiency

• Developing in vitro models that recapitulate marmoset-specific aspects of protein synthesis regulation

What are the implications of using partial versus full-length EEF2 in experimental designs?

The decision to use partial versus full-length recombinant EEF2 has significant experimental implications:

Researchers should select partial or full-length EEF2 based on their specific experimental questions, with partial constructs being more suitable for domain-specific studies and full-length proteins being necessary for comprehensive functional analyses.

How does Callithrix jacchus EEF2 compare to human EEF2, and what are the implications for translational research?

While the search results don't provide direct comparison data, EEF2 is generally highly conserved across mammals due to its essential role in protein synthesis. Any differences between marmoset and human EEF2 could have important implications for using marmosets as models for human diseases, particularly those involving translational dysregulation.

Key considerations include:

• Sequence homology analysis to identify conserved and divergent regions

• Functional assays comparing activity of both proteins under identical conditions

• Analysis of regulatory modification sites that might differ between species

• Investigation of species-specific interaction partners

What are common challenges in expressing recombinant Callithrix jacchus EEF2, and how can they be addressed?

Expression and purification of recombinant EEF2 may present several challenges:

• Inclusion body formation: EEF2 is a large protein that may form inclusion bodies in bacterial expression systems

  • Solution: Optimize expression conditions (lower temperature, reduced inducer concentration)

  • Consider solubility tags or fusion partners

  • Explore refolding protocols if necessary

• Proteolytic degradation: Partial degradation during expression or purification

  • Solution: Include protease inhibitors during purification

  • Optimize purification speed to minimize exposure time

  • Consider expression hosts with reduced protease activity

• Low yield of functional protein:

  • Solution: Screen multiple expression constructs with different boundaries

  • Test various expression hosts and conditions

  • Optimize codon usage for the expression system

• Aggregation during storage:

  • Solution: Identify optimal buffer conditions for stability

  • Consider the addition of stabilizing agents (glycerol, reducing agents)

  • Determine appropriate storage temperature and aliquoting strategy

How should researchers interpret conflicting results between in vitro studies with recombinant EEF2 and in vivo observations?

When confronted with discrepancies between in vitro and in vivo results:

• Consider differences in experimental conditions:

  • The presence of regulatory factors in vivo that may be absent in vitro

  • Differences in post-translational modifications

  • The influence of cellular compartmentalization

• Evaluate the recombinant protein's properties:

  • Confirm that the recombinant protein maintains proper folding and activity

  • Assess whether the partial protein contains all domains necessary for the observed function

  • Verify that experimental conditions support proper protein function

• Reconciliation approaches:

  • Progressively increase system complexity (from purified components to cell extracts to cellular systems)

  • Use complementary methodologies to validate observations

  • Consider species-specific factors that might influence results

What are the best storage conditions to maintain recombinant Callithrix jacchus EEF2 stability?

To maximize stability of recombinant EEF2:

• Buffer composition:

  • Use buffers with appropriate pH (typically 7.0-8.0)

  • Include stabilizing agents like glycerol (10-20%)

  • Consider the addition of reducing agents to prevent oxidation

  • Test the effect of specific ions (Mg2+, K+) that might enhance stability

• Storage practices:

  • Store concentrated stock solutions (>1 mg/ml when possible)

  • Prepare small single-use aliquots to avoid freeze-thaw cycles

  • Store at -80°C for long-term storage

  • Validate activity after various storage durations to establish stability timeline

How can researchers effectively incorporate recombinant Callithrix jacchus EEF2 into cell-free translation systems?

Cell-free translation systems provide a controlled environment for studying EEF2 function. For optimal incorporation:

• System selection:

  • Consider marmoset-derived cell extracts for species-specific studies

  • Standard rabbit reticulocyte lysate or wheat germ extract systems may be suitable for general functional studies

• Implementation protocol:

  • Deplete endogenous EEF2 from the extract when possible

  • Titrate recombinant EEF2 to determine optimal concentration

  • Include necessary cofactors (GTP, Mg2+) at appropriate concentrations

  • Monitor translation efficiency through reporter systems

• Experimental considerations:

  • Control for the influence of tags or fusion partners

  • Validate activity with known EEF2-dependent translation assays

  • Consider the impact of other translation factors that may interact with EEF2

What experimental approaches are suitable for studying EEF2 phosphorylation and its effects on translation in marmoset models?

EEF2 is regulated by phosphorylation, which typically inhibits its activity. To study this regulatory mechanism:

• In vitro approaches:

  • Reconstituted kinase assays with EEF2 kinase

  • Mass spectrometry to identify and quantify phosphorylation sites

  • Translation assays comparing effects of phosphorylated versus non-phosphorylated EEF2

• Cellular approaches:

  • Develop phospho-specific antibodies for marmoset EEF2

  • Use phosphomimetic mutations (S→D, T→E) to study functional effects

  • Apply selective inhibitors of EEF2 kinase to modulate phosphorylation

• Analytical considerations:

  • Distinguish between different phosphorylation sites that may have distinct effects

  • Consider the temporal dynamics of phosphorylation/dephosphorylation

  • Evaluate the influence of cellular stress conditions on EEF2 phosphorylation status

How can researchers design experiments to study the role of EEF2 in animal models of disease using marmosets?

Given that marmosets are used as models for various diseases , including those with potential translation dysregulation components, researchers might design experiments to:

• For infectious disease models (such as Marburg virus infection ):

  • Examine EEF2 status during different stages of infection

  • Investigate pathogen-mediated effects on translation

  • Explore EEF2-targeted interventions to modulate host response

• For neurological studies:

  • Leverage the established marmoset models for brain function research

  • Study regional differences in EEF2 activity or regulation

  • Correlate EEF2 status with pathological findings

• Experimental approaches could include:

  • Tissue-specific analysis of EEF2 modification status

  • Ex vivo translation assays from isolated tissues

  • Pharmacological manipulation of EEF2 activity in vivo