Effect of Improved pET Plasmids on Protein Expression Yield and Basal Expression

High-yield recombinant protein expression is a critical requirement in biopharmaceutical development. The pET vector family is among the most widely used systems for protein production in Escherichia coli. Shilling et al. (2020) reported that insertion of a GAGA sequence within the T7lac promoter region of pET28a enhanced protein expression and suggested broad applicability across the pET plasmid family [1]. In this study, the GAGA sequence was introduced into pET16b and pET11a plasmids, and the expression performance of the original and modified vectors was evaluated using two recombinant proteins in BL21(DE3) and BL21(DE3) pLysS cells. The modified plasmids produced variable protein yield, depending on the vector–protein combination. However, elevated basal (leaky) expression was observed in BL21(DE3) cells carrying the modified plasmids. This undesired effect was eliminated in BL21(DE3) pLysS cells. These results indicate that the impact of GAGA modification is dependent on the plasmid backbone, target protein, and host strain. While the modification can improve expression yields, we cannot say it is universally beneficial across all pET-based expression systems.