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.
journal_name
Mol Celljournal_title
Molecular cellauthors
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 SJdoi
10.1016/j.molcel.2016.06.012subject
Has Abstractpub_date
2016-07-21 00:00:00pages
337-346issue
2eissn
1097-2765issn
1097-4164pii
S1097-2765(16)30243-Xjournal_volume
63pub_type
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