Shilling, P. J. et al. Commun. Biol. 3, 214 (2020).
The pET expression plasmids are among the most popular plasmids for high levels of recombinant protein expression in Escherichia coli. These were originally designed more than 30 years ago, and even though the original design has been expanded to 103 unique plasmids, the transcription and translation control elements have remained the same. Shilling et al. have revisited the design of the pET28a plasmid, the most popular of the series. They first added the full T7 promoter sequence, which had originally been truncated upon insertion of the lac operator. Restoring the full T7 consensus sequence conferred a threefold increase in protein production. Next, they synthetically evolved the translation initiation region (TIR) which had originally formed by ad hoc genetic fusion. By screening for optimal ribosome binding, they were able to increase protein production up to 47-fold. In combination, these two strategic changes give a 33- to 121-fold increase. This approach is easy to incorporate and can be used with the other pET expression plasmids as well.
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Singh, A. Design of improved pET expression plasmids. Nat Methods 17, 655 (2020). https://doi.org/10.1038/s41592-020-0904-y