The pET series of expression plasmids are widely used for recombinant protein production in Escherichia coli. The genetic modules controlling transcription and translation in these plasmids were first described in the 1980s and have not changed since. Herein we report design flaws in these genetic modules. We present improved designs and demonstrate that, when incorporated into pET28a, they support increases in protein production. The improved designs are applicable to most of the 103 vectors in the pET series and can be easily implemented. Patrick Shilling et al. increase the protein production yield from the pET28a expression plasmid by modifying the genetic modules that control transcription and translation initiation. These improved designs are applicable to most vectors in the pET series and can be easily implemented.