Recombinant Human Elongation factor 1-gamma (EEF1G)

What is Elongation factor 1-gamma (EEF1G) and what are its primary functions?

Elongation factor 1-gamma (EEF1G) is a 437 amino acid protein that functions as a subunit of the eEF1B complex in the eukaryotic protein translation machinery. Its primary role appears to be anchoring the eEF1 complex to other cellular components . The protein contains specific structural domains that facilitate its interactions with other translation factors and cellular structures. Beyond its canonical role in translation, EEF1G has been implicated in viral protein translation in a strain-specific manner . Research indicates that EEF1G provides stability to other subunits of the eEF1B complex, as evidenced by reduced expression of eEF1B2 and eEF1D when EEF1G expression is decreased . This suggests EEF1G plays a structural role in maintaining the integrity of the entire eEF1B complex.

How is recombinant EEF1G typically produced for research applications?

Recombinant Human Elongation factor 1-gamma is typically produced using bacterial expression systems, particularly Escherichia coli. The full-length protein (1-437 amino acids) is expressed with fusion tags to facilitate purification . For example, commercial recombinant EEF1G often includes a His-tag (HHHHHH) at the N-terminus followed by a cleavage site . After expression, the protein undergoes purification processes, commonly involving affinity chromatography, to achieve high purity (>95%) suitable for research applications. The purified protein is then validated using techniques such as SDS-PAGE to confirm its molecular weight and purity . Researchers should verify that recombinant EEF1G maintains its native conformation and biological activity through functional assays before using it in their experiments.

What detection methods are available for EEF1G in research samples?

Several methods are available for detecting EEF1G in research samples:

• ELISA (Enzyme-Linked Immunosorbent Assay): Sandwich ELISA using capture and detection antibodies provides high sensitivity and specificity for quantifying EEF1G in human serum, plasma, biological fluids, and cell culture supernatants . These assays typically utilize the indirect sandwich method with double antibodies to ensure accuracy.

• Western Blotting: Western blot analysis using monoclonal or polyclonal antibodies against EEF1G is widely used to detect protein expression levels in cell or tissue lysates . This method allows visualization of both full-length and truncated forms of the protein.

• Immunoprecipitation: Used to isolate EEF1G from complex mixtures for subsequent analysis or to study protein-protein interactions.

• Mass Spectrometry: Provides detailed information about protein modifications, sequence variations, and precise quantification.

When selecting a detection method, researchers should consider factors such as required sensitivity, available sample volume, and whether qualitative or quantitative data is needed.

How does EEF1G contribute to viral replication mechanisms?

EEF1G plays a critical role in viral replication processes, particularly for HIV-1. Research has demonstrated that EEF1G is essential for HIV-1 reverse transcription, with experimental evidence showing that depletion of EEF1G by 70-90% results in complete loss of reverse transcription stimulatory activity in vitro . This effect is specific, as depletion of other translation factors like eIF3A does not show the same impact .

The mechanism involves direct association of EEF1G with viral reverse transcription complexes (RTCs) in infected cells. Isopycnographic analysis of cell lysates from HIV-1 infected cells shows co-localization of EEF1G with viral DNA and reverse transcriptase in fractions containing viral RTCs . This association appears to be mediated through interactions with the RT p51 subunit and integrase (IN) .

Additionally, strain-specific contributions of EEF1G to viral protein translation have been observed, suggesting that EEF1G may represent a host factor that influences viral tropism and replication efficiency in different cell types . The dual role of EEF1G in both reverse transcription and translation makes it a significant factor in understanding viral replication mechanisms and potentially developing antiviral strategies.

What is the relationship between EEF1G and other subunits of the eEF1B complex?

EEF1G appears to play a crucial structural role in maintaining the stability of the eEF1B complex. Experimental evidence from CRISPR/Cas9-mediated reduction of EEF1G expression demonstrates that decreased EEF1G levels lead to corresponding reductions in eEF1B2 and eEF1D expression . This suggests that EEF1G provides structural support that maintains the integrity of these other subunits.

The functional eEF1B complex consists of multiple subunits working cooperatively during protein translation. While EEF1G anchors the complex to cellular components, the other subunits perform complementary functions:

• eEF1B2 (also known as eEF1Bα): Functions as a guanine nucleotide exchange factor

• eEF1D (also known as eEF1Bδ): Also assists in guanine nucleotide exchange

• eEF1A: Delivers aminoacyl-tRNAs to the ribosome

How is EEF1G involved in cancer cell proliferation and survival?

EEF1G appears to be essential for proliferation and survival in human cancer cell lines, as evidenced by the inability to generate complete EEF1G knockout cell lines using CRISPR/Cas9 technology . Attempts to create such knockouts yielded only cells with partial reduction in EEF1G expression or expression of truncated forms of the protein, suggesting that complete loss of EEF1G function may be lethal to cancer cells .

The mechanism by which EEF1G supports cancer cell survival likely involves:

• Maintenance of protein synthesis: As a component of the translation machinery, EEF1G ensures efficient protein production needed for rapid cancer cell proliferation.

• Regulation of specific mRNA translation: EEF1G may preferentially affect translation of mRNAs encoding proteins involved in cell survival and proliferation.

• Extra-translational functions: Beyond its role in translation, EEF1G may participate in other cellular processes important for cancer cell survival.

This relationship makes EEF1G a potential target for cancer therapeutics, particularly in contexts where cancer cells show upregulated EEF1G expression compared to normal tissues. Further research is needed to fully characterize the oncogenic dependencies on EEF1G across different cancer types.

What are the key considerations for designing experiments to study EEF1G function?

When designing experiments to study EEF1G function, researchers should consider several key factors:

• Model system selection: Choose cell lines or animal models that express EEF1G at levels relevant to the research question. Consider that complete knockout of EEF1G may be lethal in some cell types .

• Expression modulation approaches:

  • For reduction of expression: Consider CRISPR/Cas9 with carefully designed gRNAs targeting exonic regions (e.g., exons six or seven ), siRNA, or shRNA approaches.

  • For rescue experiments: Design expression constructs with synonymous mutations that render the transcript resistant to the targeting mechanism (as demonstrated with gRNA1-resistant EEF1G ).

• Detection methods: Employ appropriate antibodies for Western blotting, immunoprecipitation, or immunofluorescence that recognize the specific domains of interest in EEF1G.

• Functional readouts: Select assays that measure the biological processes of interest, such as:

  • Translation efficiency (e.g., polysome profiling, ribosome profiling)

  • Viral replication (for studies on EEF1G's role in viral life cycles)

  • Cell proliferation and survival metrics

• Controls: Include appropriate positive and negative controls, such as:

  • Wild-type cells alongside modified cells

  • Rescue experiments with exogenous EEF1G expression

  • Depletion of other translation factors to demonstrate specificity

These considerations ensure that experiments yield reliable and interpretable results about EEF1G function.

How can researchers effectively manipulate EEF1G expression for functional studies?

Effective manipulation of EEF1G expression requires careful consideration of both the degree of suppression desired and the potential cellular consequences. Based on published approaches, the following methodologies can be employed:

• CRISPR/Cas9-mediated gene editing:

  • Target specific exons (such as exons six or seven) with carefully designed gRNAs

  • Create hypomorphic alleles rather than complete knockouts, as complete loss may be lethal

  • Design and validate gRNAs to minimize off-target effects

  • Example gRNA sequences that have been effective:

    • gRNA1: 5'-TGGTATTGGGAAAGGCCTGG-3' (targeting exon six)

    • gRNA3: 5'-CCTTCGCTCACCTGCCCAAG-3' (targeting exon seven)

• RNA interference:

  • siRNA for transient knockdown

  • shRNA for stable knockdown

  • Validate knockdown efficiency by Western blot and qRT-PCR

• Rescue experiments:

  • Generate expression constructs with synonymous mutations that resist targeting by the knockdown method

  • Example: Create gRNA1-resistant EEF1G with seven synonymous nucleotide mutations

  • Clone into appropriate expression vectors (e.g., pcDNA3.1) and select stable transformants

• Overexpression studies:

  • Use expression vectors with strong promoters

  • Consider inducible expression systems to control timing and level of expression

  • Tag with epitope tags (e.g., FLAG, HA) for easy detection while ensuring tags don't interfere with function

For all approaches, validation of manipulated expression levels is essential through methods such as Western blotting, with consideration of the effects on other eEF1B complex subunits (eEF1B2, eEF1D).

What purification methods yield the highest quality recombinant EEF1G for in vitro studies?

Obtaining high-quality recombinant EEF1G for in vitro studies requires optimization of expression and purification protocols. Based on established methods, the following approach yields recombinant EEF1G with >95% purity:

• Expression system:

  • Escherichia coli is the preferred system for full-length human EEF1G (1-437 amino acids)

  • Use BL21(DE3) or similar strains optimized for protein expression

  • Clone EEF1G into vectors with N-terminal tags (e.g., His-tag) to facilitate purification

• Induction conditions:

  • Optimize temperature (typically 16-30°C), inducer concentration (IPTG), and induction time

  • Lower temperatures (16-18°C) often yield more soluble protein

  • Consider auto-induction media for high-density cultures

• Lysis and initial clarification:

  • Use buffer systems compatible with downstream applications

  • Include protease inhibitors to prevent degradation

  • Employ sonication or high-pressure homogenization for efficient lysis

  • Remove insoluble material by centrifugation at >20,000g

• Affinity chromatography:

  • For His-tagged EEF1G, use Ni-NTA or IMAC columns

  • Apply gradual imidazole gradients for elution to separate full-length protein from truncated products

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and ensure monodispersity

  • Ion exchange chromatography for removing contaminants with different charge properties

• Quality control:

  • SDS-PAGE to verify molecular weight (~50 kDa) and purity (>95%)

  • Mass spectrometry to confirm protein identity

  • Dynamic light scattering to assess homogeneity

  • Functional assays to confirm biological activity

These purification methods ensure that the recombinant EEF1G maintains its native conformation and activity, making it suitable for biochemical and structural studies.

How should researchers interpret changes in EEF1G expression in relation to other eEF1 complex members?

When analyzing changes in EEF1G expression, researchers should carefully consider the interdependent relationship with other eEF1 complex members. Experimental evidence indicates that reduced EEF1G expression leads to corresponding decreases in eEF1B2 and eEF1D levels , suggesting a coordinated regulation of these subunits. When interpreting such data:

• Assess proportional changes: Determine whether the reduction in eEF1B2 and eEF1D is proportional to EEF1G reduction or follows a different pattern, which may indicate specific regulatory mechanisms.

• Consider stability relationships: Evaluate whether the observed changes reflect direct stability effects (EEF1G stabilizing other subunits) or indirect effects through altered gene expression or protein synthesis.

• Examine functional consequences: Analyze how changes in the entire complex affect cellular processes such as:

  • Global translation rates

  • Translation of specific mRNAs

  • Cell growth and proliferation

• Control for compensatory mechanisms: Monitor potential upregulation of other translation factors that might compensate for reduced eEF1 complex function.

A comprehensive analysis should include quantification of all complex subunits using methods such as Western blotting with appropriate normalization, accompanied by functional assays to determine the impact on translation efficiency.

What are the key considerations when analyzing EEF1G's role in viral infection studies?

When analyzing EEF1G's role in viral infection studies, researchers should consider several key factors to ensure accurate interpretation of results:

• Virus strain specificity: Research indicates that EEF1G contributes to viral protein translation in a strain-specific manner . Therefore, observations from one viral strain may not generalize to others. Researchers should:

  • Test multiple viral strains/isolates

  • Compare results across different viral families

  • Consider evolutionary relationships between tested viruses

• Distinguishing direct vs. indirect effects: EEF1G influences both translation and other cellular processes. When analyzing its role in viral infection:

  • Separate effects on viral genome replication from effects on viral protein translation

  • Use time-course experiments to determine when EEF1G is most critical

  • Design controls that distinguish general translation effects from virus-specific effects

• Integration with other host factors: EEF1G works in concert with other host factors during viral replication. Analysis should:

  • Consider interactions with known virus-interacting host proteins

  • Evaluate whether EEF1G effects are dependent on specific cellular conditions

  • Examine potential redundancy or synergy with other translation factors

• Quantitative assessment of viral replication stages: For HIV-1 specifically, researchers should quantify:

  • Early and late reverse transcription products (as EEF1G depletion causes complete loss of reverse transcription stimulatory activity)

  • Integration events

  • Viral protein synthesis

  • Virus particle production

By systematically addressing these considerations, researchers can more accurately define EEF1G's specific contributions to viral replication cycles.

How can researchers differentiate between EEF1G's canonical translation functions and its potential non-canonical roles?

Differentiating between EEF1G's canonical translation functions and its non-canonical roles requires strategic experimental approaches:

• Domain-specific mutational analysis:

  • Generate EEF1G variants with mutations in domains involved in eEF1 complex formation versus domains potentially involved in other functions

  • Assay each variant for specific activities to map structure-function relationships

  • Compare effects on global translation versus specific cellular processes

• Temporal separation of functions:

  • Use rapid inducible depletion systems (e.g., auxin-inducible degron tags) to distinguish immediate effects (likely translation-related) from delayed effects (potentially non-canonical)

  • Conduct time-course experiments following EEF1G depletion or overexpression

• Comparative analysis with other translation factors:

  • Compare phenotypes resulting from EEF1G manipulation versus manipulation of other eEF1 complex members

  • Effects unique to EEF1G may represent non-canonical functions

• Interaction network analysis:

  • Perform comprehensive protein-protein interaction studies (e.g., BioID, IP-MS)

  • Map EEF1G interactions in different cellular compartments

  • Identify interaction partners not involved in translation

• Context-specific function assessment:

  • Evaluate EEF1G functions under various cellular conditions (stress, differentiation, etc.)

  • Analyze EEF1G's role in specific cellular compartments through fractionation studies

For instance, in HIV-1 studies, researchers have distinguished EEF1G's direct role in reverse transcription from its translation functions by demonstrating its physical association with reverse transcription complexes (RTCs) in density gradient fractions that contain viral DNA and RT , representing a clear non-canonical function.

What are the most promising future research directions for understanding EEF1G function?

Based on current knowledge, several promising research directions for EEF1G emerge:

• Structural biology approaches to determine the three-dimensional structure of EEF1G alone and in complex with its binding partners, providing insights into its multiple functional roles. This would help elucidate how EEF1G anchors the eEF1B complex to cellular components and interacts with viral proteins like HIV-1 reverse transcriptase .

• Systems biology studies to comprehensively map EEF1G's interaction network across different cellular conditions and disease states. This would reveal context-specific functions and potential regulatory mechanisms.

• Translatomics research to identify mRNAs whose translation is specifically affected by EEF1G modulation, potentially revealing specialized roles in regulating expression of specific gene sets.

• Therapeutic targeting approaches exploring EEF1G as an antiviral target, given its essential role in HIV-1 reverse transcription . Compounds that disrupt the interaction between EEF1G and viral components without affecting essential cellular functions could represent novel antiviral strategies.

• Cancer biology investigations to further characterize EEF1G's role in cancer cell survival and proliferation , including potential as a biomarker or therapeutic target in specific cancer types.

• Post-translational modification mapping to identify how EEF1G function is regulated through modifications such as phosphorylation, which could reveal mechanisms for dynamically controlling its various functions.

These research directions would significantly advance our understanding of EEF1G beyond its canonical role in translation, potentially leading to new therapeutic approaches for viral infections and cancer.

What methodological advances would most benefit EEF1G research?

Methodological advances in several areas would significantly benefit EEF1G research:

• Improved genetic manipulation techniques that allow for conditional and tissue-specific modulation of EEF1G expression. Current evidence suggests complete EEF1G knockout may be lethal , so refined approaches such as:

  • Inducible degradation systems

  • Domain-specific interference methods

  • Tissue-specific conditional knockouts
    would enable more nuanced functional studies.

• Advanced structural biology methods to overcome challenges in determining the structure of the complete eEF1 complex:

  • Cryo-electron microscopy for visualizing dynamic complexes

  • Integrative structural biology approaches combining multiple techniques

  • In-cell structural determination methods

• Single-molecule techniques to study EEF1G's dynamic interactions during translation:

  • Single-molecule FRET to observe conformational changes

  • Live-cell single-molecule tracking to monitor dynamics in real time

  • Optical tweezers to measure forces involved in translation processes

• Proteomics advances for studying EEF1G interactions and modifications:

  • Proximity labeling methods with improved spatial resolution

  • Mass spectrometry techniques with enhanced sensitivity for post-translational modifications

  • Targeted proteomics approaches for quantifying low-abundance complexes

• Computational approaches for predicting:

  • Protein-protein interaction interfaces

  • Drug binding sites for targeting EEF1G-virus interactions

  • Functional consequences of naturally occurring variants