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 m

SUBMITTER: Goldenzweig A 

PROVIDER: S-EPMC4961223 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature