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

Abstract

SummaryUpon 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.

Reducing proteolytic liability of a MMP‐14 inhibitory antibody by site‐saturation mutagenesis

“…44 cdMMP-12 mutant was designed and produced following the PROSS algorithm. 46 The inhibitory functions of purified Fab 3A2 wild-type and mutants on MMP-2, cdMMP-9/-12/-14 proteolytic activities were measured by FRET assays. 27 Broad spectrum inhibitor GM6001 (EMD) was used to titrate MMP concentrations.…”

Design of a basigin‐mimicking inhibitor targeting the malaria invasion protein RH5

“…The resulting S i, j values were then transformed to fold-enrichment values by: We refined the RH5-bound basigin structure (PDB entry: 4U0Q) by four iterations of side-chain packing and side-chain and backbone minimization. 27 The experimental mutation tolerance map was used to define a sequence space with all beneficial mutations, followed by a combinatorial sequence optimization. Starting from the refined structure, we imposed coordinate restraints relative to the coordinates in the PDB entry and implemented four iterations of sequence design, sidechain, and backbone minimization, while alternating soft and hard repulsive potentials.…”

“…Starting from the refined structure, we imposed coordinate restraints relative to the coordinates in the PDB entry and implemented four iterations of sequence design, sidechain, and backbone minimization, while alternating soft and hard repulsive potentials. 27 The energy difference between the refined structure and the optimized highaffinity mutant was calculated using the talaris2014 energy function. 28 2.8 | BSG and BSG des7 production and purification BSG and BSG des7 were transformed into pet21 H-Tev plasmid by RF cloning.…”

Overlay-independent comparisons of X-ray structures reveal small, systematic conformational changes in liganded acetylcholinesterase

“…Recently the first high yield bacterial expression of a correctly folded, catalytically functional AChE was achieved (10). The 51 simultaneous mutations created by automated protein sequence design were necessary, resulting in expression of a non-glycosylated hAChE mutant (10).…”

“…The 51 simultaneous mutations created by automated protein sequence design were necessary, resulting in expression of a non-glycosylated hAChE mutant (10). The readily determined X-ray 3D structure of this mutant allows for analysis on degree of structural similarity of its backbone fold with unliganded (Apo) wt hAChE and nature of driving forces that led to "self-stabilization" of this protein during its expression in bacteria, even in the absence of N-linked polysaccharide chains.…”