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

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

Author information

1
Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
2
School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.
3
Department of Life Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 8410501, Israel.
4
Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel; Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 7610001, Israel.
5
Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 7610001, Israel.
6
Bioinformatics & Biological Computing Unit, Weizmann Institute of Science, Rehovot 7610001, Israel.
7
Department of Neurobiology, Weizmann Institute of Science, Rehovot 7610001, Israel.
8
Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address: dan.tawfik@weizmann.ac.il.
9
Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address: sarel@weizmann.ac.il.

Abstract

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 http://pross.weizmann.ac.il.

PMID:
27425410
PMCID:
PMC4961223
DOI:
10.1016/j.molcel.2016.06.012
[Indexed for MEDLINE]
Free PMC Article

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