Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

My pET project: Improving plasmid design for increased protein production (#314)

Patrick J Shilling 1 2 , Kiavash Mirzadeh 1 3 , Alister J Cumming 1 , Magnus Widesheim 1 , Zoe Köck 1 , Daniel O Daley 1 3
  1. Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
  2. The University of Melbourne, Parkville, VICTORIA, Australia
  3. CloneOpt AB, Stockholm, Sweden

Recombinant expression systems are routinely used by researchers the world over. Genes are pasted into any desired plasmid in the hope that production will reach yields suitable for subsequent experiments. Maximising production levels, thereby facilitating downstream processing is usually the goal. However, it is typical that yields are marginal at best. It may surprise many that the most popular workhorse plasmids, those in the pET series, have largely remained unchanged since the 1980s. How tuned or efficient are these plasmids for protein expression? In a succession of experiments we identified pET28a, the most widely used expression plasmid of the series, displayed suboptimal expression1,2.

Our investigation focused on modules controlling transcription and translation initiation (TI). We identified that the T7 transcriptional promoter (T7p) in 85 of 103 available pET plasmids, is a truncated variant. When corrected, production increased three-fold.

Translation initiation is the rate limiting step during protein synthesis. TI is dependent on the nucleotide sequence of the translation initiation region (TIR). The TIR stretches from the Shine-Dalgarno up to codon five of the open reading frame. For E. coli mRNAs, ad hoc cloning produces novel TIRs that may not be efficiently recognised by host cell ribosomes. Centring on the start codon, we employed nucleotide randomisation and selection of optimal TIRs. The directed-evolution approach identified a new TIR-sequence that improved protein expression by 47-fold. Combined with the T7p correction, we noted a 121-fold improvement.

Our results indicate that pET plasmids and ad hoc cloning can result in inefficient protein expression in E. coli. This work has received considerable interest, with 10,000 paper accesses from May-Oct 20201, many requests for plasmid material, and notation as a ‘research highlight’ in Nature Methods3. We believe limited protein expression may be overcome for many protein targets when incorporating the T7p correction and the directed-evolution technique.

  1. Shilling, P. J. et al. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol 3, 1–8 (2020).
  2. Mirzadeh, K. et al. Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microbial Cell Factories 19, 85 (2020).
  3. Singh, A. Design of improved pET expression plasmids. Nature Methods 17, 655–655 (2020).