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

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. [Display omitted] •A new computational method is used to stabilize five recalcitrant proteins•Designed variants show higher expression and stability with unmodified function•A designed human acetylcholinesterase variant expresses solubly in bacteria•The method is fully automated and implemented on a webserver Heterologous expression of proteins and their mutants often results in misfolding and aggregation. Goldenzweig et al. (2016) developed an automated algorithm for protein stabilization requiring minimal experimental testing; for instance, the five tested variants of human acetylcholinesterase showed ≥100-fold higher soluble bacterial expression and higher melting temperatures than wild-type.