Identification of an alternative translation initiation site in the sequence of the commonly used Glutathione S-Transferase tag.

Research paper by Sarah C SC Bernier, Louis-Philippe LP Morency, Rafael R Najmanovich, Christian C Salesse

Indexed on: 10 Sep '18Published on: 10 Sep '18Published in: Journal of biotechnology


A large number of proteins are expressed in fusion with a tag to perform their purification. Glutathione-S-Transferase (GST) is a widely used tag to achieve passenger protein purification. Accordingly, commonly used commercial expression vectors contain the coding sequence of GST in order to express fusion proteins. However, fusion proteins are sometimes expressed in a truncated form that may result from an incomplete synthesis, proteolytic cleavage or the presence of a functional alternative translation initiation site. In particular, a truncated as well as a full-length fusion protein were observed when expressing RGS9-1 Anchor protein without its C-terminal segment (bR9AP) in fusion with GST. Moreover, this truncated protein was found to be purified together with the full-length fusion protein. Here, we identified for the first time an alternative translation initiation site within the sequence of GST that likely becomes accessible for translation only when it is fused with a passenger protein. Indeed, bioinformatics analyses suggest that the secondary structure of the mRNA of the GST-bR9AP fusion protein is different from that of GST alone, which likely allows accessibility of an alternative Shine-Dalgarno sequence coupled with an additional initiation codon within the sequence of GST. The functionality of this alternative translation initiation site was confirmed by site-directed mutagenesis, which resulted in the absence of a truncated fusion protein and, consequently, only a purified full-length fusion protein. This is an extremely important finding in view of the wide use of GST as a purification and solubility-enhancing tag. Copyright © 2018. Published by Elsevier B.V.