Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability

Mol Cell. 2016 Jul 21;63(2):337-346. doi: 10.1016/j.molcel.2016.06.012. Epub 2016 Jul 14.


Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at

MeSH terms

  • Acetylcholinesterase / chemistry
  • Acetylcholinesterase / genetics
  • Acetylcholinesterase / metabolism*
  • Algorithms
  • Automation, Laboratory
  • Computational Biology / methods*
  • Computer Simulation
  • Computer-Aided Design
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA (Cytosine-5-)-Methyltransferases / metabolism
  • DNA Methyltransferase 3A
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • GPI-Linked Proteins / chemistry
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / metabolism
  • Gene Expression Regulation, Bacterial
  • Gene Expression Regulation, Enzymologic
  • Mutation
  • Phosphoric Triester Hydrolases / genetics
  • Phosphoric Triester Hydrolases / metabolism
  • Protein Conformation
  • Protein Denaturation
  • Protein Engineering / methods*
  • Protein Stability
  • Sirtuins / genetics
  • Sirtuins / metabolism
  • Structure-Activity Relationship
  • Temperature


  • DNMT3A protein, human
  • GPI-Linked Proteins
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA Methyltransferase 3A
  • ACHE protein, human
  • Acetylcholinesterase
  • Phosphoric Triester Hydrolases
  • SIRT6 protein, human
  • Sirtuins