title: Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.
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.
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Adi
authors.initials: A
authors.lastname: Goldenzweig
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Moshe
authors.initials: M
authors.lastname: Goldsmith
authors.affiliation: School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.
authors.forename: Shannon E
authors.initials: SE
authors.lastname: Hill
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 8410501, Israel.
authors.forename: Or
authors.initials: O
authors.lastname: Gertman
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Paola
authors.initials: P
authors.lastname: Laurino
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel; Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Yacov
authors.initials: Y
authors.lastname: Ashani
authors.affiliation: Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Orly
authors.initials: O
authors.lastname: Dym
authors.affiliation: Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Tamar
authors.initials: T
authors.lastname: Unger
authors.affiliation: Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Shira
authors.initials: S
authors.lastname: Albeck
authors.affiliation: Bioinformatics & Biological Computing Unit, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Jaime
authors.initials: J
authors.lastname: Prilusky
authors.affiliation: School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.
authors.forename: Raquel L
authors.initials: RL
authors.lastname: Lieberman
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 8410501, Israel.
authors.forename: Amir
authors.initials: A
authors.lastname: Aharoni
authors.affiliation: Department of Neurobiology, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Israel
authors.initials: I
authors.lastname: Silman
authors.affiliation: Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Joel L
authors.initials: JL
authors.lastname: Sussman
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address: dan.tawfik@weizmann.ac.il.
authors.forename: Dan S
authors.initials: DS
authors.lastname: Tawfik
authors.affiliation: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address: sarel@weizmann.ac.il.
authors.forename: Sarel J
authors.initials: SJ
authors.lastname: Fleishman
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000465:Algorithms; D057205:Automation, Laboratory; D019295:Computational Biology / Q000379:methods*; D003198:Computer Simulation; D017076:Computer-Aided Design; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D000090205:DNA Methyltransferase 3A; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D009154:Mutation; D044345:Phosphoric Triester Hydrolases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D037761:Sirtuins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: C000717967:DNMT3A protein, human; D058851:GPI-Linked Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C548244:ACHE protein, human; D000110:Acetylcholinesterase; D044345:Phosphoric Triester Hydrolases; C516592:SIRT6 protein, human; D037761:Sirtuins