Abstract
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.
Highlights
The pET series of expression plasmids are widely used for recombinant protein production in Escherichia coli
The nucleotide sequence is derived from the consensus φ10 promoter in the T7lac φ10 (T7) phage, which is 23 nucleotides long and sits −17 to +6 relative to the messenger RNA start site (Fig. 1b)[6]
We noted that pET28a only contains the −17 to +2 region, as four nucleotides were removed when the lac operator sequence was originally introduced in the early generation pET plasmids[5]
Summary
The pET series of expression plasmids are widely used for recombinant protein production in Escherichia coli. We present improved designs and demonstrate that, when incorporated into pET28a, they support increases in protein production. Novagen and Invitrogen subsequently expanded the series to 103 unique expression plasmids These expression plasmids support high levels of transcription in strains of Escherichia coli that contain a lysogenised DE3 phage fragment encoding the T7 RNA polymerase and they have become a workhorse for the scientific community[3,4]. To date, they have been described in >220,000 published research studies (>12,000 per year; Supplementary Fig. 1). The study describes an implementable strategy for increasing recombinant protein yields using pET expression plasmids
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