Optimized purification workflow for advanced eEF1A studies: Impact of His-tagging on stability, functionality, and...

Area of Science:

• Molecular Biology
• Protein Biochemistry

Background:

• Eukaryotic elongation factor 1A (eEF1A) is essential for protein translation.
• Its bacterial homolog, EF-Tu, is well-characterized, but eEF1A's functions are less understood due to purification challenges.

Purpose of the Study:

• To optimize an affinity chromatography protocol for Saccharomyces cerevisiae eEF1A.
• To evaluate the impact of N- and C-terminal His-tagging on eEF1A stability, functionality, and purification yield.

Main Methods:

• Affinity chromatography was used to purify eEF1A.
• N- and C-terminal His-tagged eEF1A variants were constructed and analyzed.
• Nano differential scanning fluorimetry (NanoDSF) assessed protein stability.
• Ternary complex formation was evaluated in vitro.

Main Results:

• An N-terminal deletion impaired ternary complex formation, but N-terminal His-tagging did not.
• NanoDSF showed N-terminal His-tag destabilizes eEF1A, while C-terminal His-tag preserves integrity and increases yield.
• Reducing glycerol concentration from 25% to 10% accelerated purification without affecting stability or tRNA binding.

Conclusions:

• A C-terminal His-tagging strategy combined with reduced glycerol concentration provides an efficient method for purifying functional eEF1A.
• This optimized protocol facilitates further research into the canonical and non-canonical roles of eEF1A.