Recombinant Cricetulus griseus Elongation factor 1-alpha 1 (EEF1A1)

What is Cricetulus griseus Elongation factor 1-alpha 1 (EEF1A1) and what are its primary functions?

Cricetulus griseus (Chinese hamster) Elongation factor 1-alpha 1 (EEF1A1) is a highly conserved protein essential for protein synthesis, specifically during the elongation phase of translation. This 54.2 kDa protein functions by delivering aminoacyl-tRNAs to the ribosome A-site in a GTP-dependent manner . Beyond translation, EEF1A1 participates in various cellular processes including cytoskeletal organization, protein degradation, and signal transduction pathways.

Research methodological considerations:

• For studying canonical translation functions, in vitro translation assays with purified components are recommended

• For investigating non-canonical functions, protein-protein interaction studies should be employed

• When interpreting results, consider that EEF1A1 has a paralog (EEF1A2) with tissue-specific expression patterns

What expression systems are most effective for producing recombinant Cricetulus griseus EEF1A1?

Based on published research, E. coli serves as an effective expression system for recombinant EEF1A1 production . The protein is typically expressed with N-terminal tags (6xHis or GST) to facilitate purification.

Research methodology recommendations:

• Express in E. coli with N-terminal 6xHis-tag for applications requiring high purity

• Use BL21(DE3) or JM109 E. coli strains for optimal expression

• Typical purification achieves >85% purity as determined by SDS-PAGE

• For functional studies requiring native conformations, consider mammalian expression systems

Storage and handling protocols:

• Store in Tris/PBS-based buffer with 5-50% glycerol at -20°C

• For lyophilized preparations, reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Complete with glycerol to 50% final concentration

• Avoid repeated freeze-thaw cycles to maintain protein integrity

How does the EEF1A1 promoter compare to the CMV promoter for stable expression in CHO cells?

The Chinese hamster EEF1A1 promoter represents a viable alternative to the widely used cytomegalovirus (CMV) promoter for recombinant protein expression in biopharmaceutical applications. Comparative studies have established several key differences:

Research data shows the EEF1A1 promoter and its surrounding DNA regions effectively maintain high-level and stable expression of recombinant proteins in CHO cells . When combined with the Epstein-Barr virus terminal repeat (EBVTR) fragment, expression stability is further enhanced, making these plasmid vectors particularly suitable for biopharmaceutical applications where long-term stability is critical .

What strategies can optimize EEF1A1-based expression vectors for improved efficiency?

Several research-validated approaches can optimize EEF1A1-based expression vectors:

What methodological approaches are most effective for studying EEF1A1 interactions with other proteins?

Several validated experimental approaches can be employed to investigate EEF1A1 protein interactions:

• Yeast two-hybrid screening: Effective for initial identification of novel interaction partners, as demonstrated in studies identifying interactions between EEF1A1 and sphingosine kinase .

• Recombinant protein production: Express EEF1A1 as a GST fusion protein in E. coli for in vitro binding assays. The coding sequence can be amplified from human foreskin fibroblast cDNA and hemagglutinin (HA) epitope-tagged at the C-terminus using the following primer strategy:

  • Forward primer: 5′-CCGGATCCGCCACCATGGGAAAGGAAAAGACTCATA-3′

  • Reverse primer (with HA tag): 5′-GGCTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTATTTAGCCTTCTGAGCTTTCTG-3′

• Co-immunoprecipitation assays: Using lysates from cells expressing FLAG-tagged interacting proteins and HA-tagged eEF1A isoforms to validate interactions in a cellular context .

• In vitro phosphorylation studies: For studying regulatory modifications, in vitro phosphorylation can be performed using 1 μg of purified GST-eEF1A1, 50 ng of kinase (e.g., S6 kinase), in buffer containing 20 mM MOPS (pH 7.2), 5 μM EDTA, 0.01% glycerol, 2 μg/ml bovine serum albumin, 1 mM ATP, 20 mM β-glycerophosphate, 1 mM dithiothreitol, 1 mM orthovanadate, and 5 mM EGTA .

How can researchers investigate the differential regulation of EEF1A1 versus EEF1A2 isoforms?

Despite high sequence homology (approximately 92% identity), EEF1A1 and EEF1A2 exhibit distinct regulatory characteristics that require specific experimental strategies:

• Isoform-specific expression analysis:

  • EEF1A1 cDNA can be amplified from human foreskin fibroblast cDNA

  • EEF1A2 cDNA can be obtained from mouse brain cDNA

• Comparative binding studies: When investigating differential protein interactions, both isoforms should be tested in parallel using identical experimental conditions. Express both proteins with the same tag (e.g., HA-tag) and position (N or C-terminal) for valid comparison .

• Post-translational modification analysis: Mass spectrometry-based approaches can identify differential patterns of phosphorylation, methylation, or other modifications between isoforms that may explain functional differences.

• Functional comparisons: Design translation elongation assays that can detect subtle differences in tRNA binding affinity, GTP hydrolysis rates, or ribosome interaction dynamics between the two isoforms.

What are the critical factors in experimental design when investigating EEF1A1 function in protein translation?

When designing experiments to study EEF1A1's role in translation, researchers should consider:

• Source material purity: Recombinant protein should achieve >85% purity by SDS-PAGE for reliable functional studies .

• Post-translational modification status: Consider whether the E. coli-expressed recombinant protein (lacking eukaryotic modifications) is suitable for your specific research question, or if expression in mammalian cells is necessary.

• Functional assay design:

  • GTP hydrolysis assays to measure the catalytic activity

  • Aminoacyl-tRNA binding assays to assess substrate interaction

  • In vitro translation systems to evaluate the impact on protein synthesis rates and fidelity

• Control experiments:

  • Include GTPase-deficient mutants as negative controls

  • Compare with EEF1A2 isoform to identify unique functional properties

  • Include appropriate buffer-only and inactive protein controls

How should researchers approach studying EEF1A1's non-canonical functions?

Beyond its role in translation, EEF1A1 participates in various cellular processes requiring specific experimental approaches:

• Cytoskeletal interactions: Actin binding and bundling assays, using purified components and fluorescence microscopy to visualize cytoskeletal organization.

• Protein quality control: Examining interactions with components of the protein degradation machinery through co-immunoprecipitation and functional assays.

• Signal transduction pathways: Investigating interactions with kinases and other signaling molecules, as demonstrated by the established interaction between EEF1A1 and sphingosine kinase .

• Experimental validation strategies:

  • RNA interference to deplete endogenous EEF1A1

  • Rescue experiments with wild-type versus mutant EEF1A1

  • Domain mapping to identify regions mediating specific interactions

How can the EEF1A1 promoter be engineered for optimized recombinant protein production?

The EEF1A1 promoter offers significant advantages for stable protein expression in CHO cells that can be further enhanced through rational engineering:

What approaches can identify critical regulatory elements within the EEF1A1 gene region?

To identify functional regulatory elements in the EEF1A1 gene region:

• Systematic deletion analysis: Creating a series of deletions in both upstream and downstream flanking regions followed by functional testing, as demonstrated in recent research .

• Reporter gene assays: Using fluorescent proteins like eGFP to quantitatively assess the impact of specific elements on expression levels and stability .

• Chromatin structure analysis: Investigating the epigenetic landscape around the EEF1A1 gene to identify regions of open chromatin that may harbor regulatory elements.

• Comparative genomics: Analyzing conservation of non-coding sequences across species to identify potentially important regulatory regions.

The research evidence indicates that upstream flanking regions contain elements that positively regulate transcription, which can be leveraged in expression vector design for biopharmaceutical applications .