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

The eukaryotic elongation factor 1A (eEF1A) is crucial for translation, delivering aminoacyl-tRNAs to the ribosomal A site. While its bacterial counterpart, elongation factor thermo unstable (EF-Tu), has been extensively studied, the canonical and non-canonical roles of eEF1A remain less understood, partly due to the lack of an efficient purification method. This study optimized an affinity chromatography-based protocol for Saccharomyces cerevisiae eEF1A, evaluating the effects of N- and C-terminal His-tagging on stability, functionality, purification, and yield. While N-terminal deletion of four residues impaired ternary complex formation in vitro, His-tagging at the same position did not. However, nano differential scanning fluorimetry (NanoDSF) revealed that an N-terminal His-tag destabilizes eEF1A, whereas a C-terminal His-tag preserves its integrity and enhances yield. Additionally, reducing glycerol concentration from 25% to 10% expedited purification without compromising stability or tRNA binding. This optimized C-terminal His-tag protocol provides a streamlined approach for studying the functional and dynamic properties of eEF1A.