Herein we constructed the pET^OPT collection by re-engineering the most commonly used expression plasmids for recombinant protein production in Escherichia coli (pET28, pET21, pET22, pET15, pET30, pET32). Each pET^OPT plasmid has been designed for strains with both high and low levels of the T7 RNA polymerase by incorporating improved genetic modules for transcriptional initiation, transcriptional termination, translation Initiation and plasmid selection / maintenance. And all plasmids are available with both the Tn903.1^MIN (kana25) and Tn3.1^MIN (amp20) antibiotic resistance markers, so that users have flexibility. pET^OPT plasmids contain the same bells and whistles as original pET expression plasmids, but give higher production titres and better plasmid maintenance. They will provide a competitive advantage to biochemists, biophysicist and structural biologists in academia, as well as in the biotech and biopharma sectors.