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

Adi Goldenzweig, Moshe Goldsmith, Shannon E. Hill, Or Gertman, Paola Laurino, Yacov Ashani, Orly Dym, Tamar Unger, Shira Albeck, Jaime Prilusky, Raquel L. Lieberman, Amir Aharoni, Israel Silman, Joel L. Sussman, Dan S. Tawfik, Sarel J. Fleishman

Research output: Contribution to journalArticlepeer-review

240 Scopus citations


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

Original languageEnglish
Pages (from-to)337-346
Number of pages10
JournalMolecular Cell
Issue number2
StatePublished - 21 Jul 2016

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability'. Together they form a unique fingerprint.

Cite this