Fleishman SJ

References (3)

Title : PROSS 2: a new server for the design of stable and highly expressed protein variants - Weinstein_2020_Bioinformatics_37_123
Author(s) : Weinstein JJ , Goldenzweig A , Hoch SY , Fleishman SJ
Ref : Bioinformatics , 37 :123 , 2020
Abstract : Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence analysis, error handling and automated analysis of the quality of the sequence alignment that is used in design calculations. PROSS2 is freely available for academic use at https://pross.weizmann.ac.il.
ESTHER : Weinstein_2020_Bioinformatics_37_123
PubMedSearch : Weinstein_2020_Bioinformatics_37_123
PubMedID: 33367682

Title : Highly active enzymes by automated combinatorial backbone assembly and sequence design - Lapidoth_2018_Nat.Commun_9_2780
Author(s) : Lapidoth G , Khersonsky O , Lipsh R , Dym O , Albeck S , Rogotner S , Fleishman SJ
Ref : Nat Commun , 9 :2780 , 2018
Abstract : Automated design of enzymes with wild-type-like catalytic properties has been a long-standing but elusive goal. Here, we present a general, automated method for enzyme design through combinatorial backbone assembly. Starting from a set of homologous yet structurally diverse enzyme structures, the method assembles new backbone combinations and uses Rosetta to optimize the amino acid sequence, while conserving key catalytic residues. We apply this method to two unrelated enzyme families with TIM-barrel folds, glycoside hydrolase 10 (GH10) xylanases and phosphotriesterase-like lactonases (PLLs), designing 43 and 34 proteins, respectively. Twenty-one GH10 and seven PLL designs are active, including designs derived from templates with <25% sequence identity. Moreover, four designs are as active as natural enzymes in these families. Atomic accuracy in a high-activity GH10 design is further confirmed by crystallographic analysis. Thus, combinatorial-backbone assembly and design may be used to generate stable, active, and structurally diverse enzymes with altered selectivity or activity.
ESTHER : Lapidoth_2018_Nat.Commun_9_2780
PubMedSearch : Lapidoth_2018_Nat.Commun_9_2780
PubMedID: 30018322

Title : Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability - Goldenzweig_2016_Mol.Cell_63_337
Author(s) : Goldenzweig A , Goldsmith M , Hill SE , Gertman O , Laurino P , Ashani Y , Dym O , Unger T , Albeck S , Prilusky J , Lieberman RL , Aharoni A , Silman I , Sussman JL , Tawfik DS , Fleishman SJ
Ref : Mol Cell , 63 :337 , 2016
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 approximately 2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20 degrees 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.
ESTHER : Goldenzweig_2016_Mol.Cell_63_337
PubMedSearch : Goldenzweig_2016_Mol.Cell_63_337
PubMedID: 27425410
Gene_locus related to this paper: human-ACHE