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 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.

Graphical abstract

Figure 1

Eliminating Potentially Destabilizing Mutations through Homologous-Sequence Analysis and Computational Mutation Scanning (A) (Left) Sequence logo for hAChE position Gly416. Letter height represents the respective amino acid’s frequency in an alignment of homologous AChE sequences. The evolutionarily “allowed” sequence space (PSSM scores ≥0) at position 416 includes the nine amino acids shown. (Right) Structural models of mutations to the evolutionarily favored amino acid His, and to Gln, which is favored by Rosetta energy calculations. The His side chain is strained due to its proximity to the bulky Tyr503 aromatic ring, whereas the Gln side chain is relaxed and forms a favorable hydrogen bond with Tyr503 (dashed line). (B) Computational mutation scanning correctly identifies all of the destabilizing mutations and ⅔ of the stabilizing mutations in yeast triosephosphate isomerase (TIM). Sullivan et al. (2012) used consensus design to predict 23 mutations that would stabilize yeast TIM and measured each mutant’s T_m difference relative to wild-type (ΔT_m). We used computational mutation scanning to predict the effects of each mutation on stability (ΔΔG_calc). Four mutations were experimentally found to be highly deleterious, resulting in no functional expression, and therefore the T_m for these could not be measured (open circles); these four mutations have highly unfavorable ΔΔG_calc values (>4.6 Rosetta energy units; R.e.u.). See also Table S1.

Figure 2

Design of a Stable hAChE variant and Its Functional Expression in Bacteria (A) The structural underpinnings of stabilization in the designed variant dAChE4. Wild-type hAChE is shown in blue and 51 mutated positions, which are distributed throughout dAChE4, are indicated by orange spheres. Thumbnails highlight stabilizing effects of selected mutations. (B) Bacterial lysate activity levels of designed AChEs normalized to hAChE activity. Crude lysates were derived from 250 ml flasks (medium scale) or 0.5 ml E. coli cultures grown in a 96-well plate (small scale). The higher activity levels in the designed variants reflect higher levels of soluble, functional enzyme. (C) Designed AChE variants (colored lines) show higher resistance to heat inactivation compared to hAChE (black). Residual activities following incubation at different temperatures were measured in bacterial lysates and normalized to the activity in nontreated lysates. (D) Sub-Ångstrom accuracy in alignment of key residues in the vicinity of the catalytic triad in the crystallographic structure of dAChE4 (PDB: 5HQ3, yellow) compared to hAChE (PDB: 4EY4, green). See also Figure S1 and Tables S2–S5.

Figure 3

Higher Expression, Stability, and Activity of Designed Variants of PTE, SIRT6, Dnmt3a, and Myoc-OLF (A) Compared to the previously engineered variant PTE-S5 (Roodveldt and Tawfik, 2005), the design dPTE2 shows higher resistance to inactivation by the metal chelator 1,10-phenanthroline (50 μM), indicating higher metal affinity and stability. (B) (Upper panel) The previously engineered E1 variant of hSIRT6 shows a 3-fold decline in in vivo expression levels, whereas following computational design, dSIRT6-E1 (denoted as dE1) recapitulates hSIRT6’s expression levels (western blot quantified using ImageJ). Actin expression levels are provided as control. (Lower panel) dE1 exhibits higher in vivo histone H3 Lys56-deacylation activity compared to hSIRT6. HY denotes a loss-of-function mutant of hSIRT6; H3 expression levels are provided as control. (C) The designed variant dDnmt3A shows 10-fold higher DNA-methylation compared to hDnmt3a as determined by the levels of incorporation of H³-methyl groups in the presence of equal enzyme concentrations. (D) dMyoc-OLF is more thermostable than hMyoc-OLF, and addition of Ca⁺² further stabilizes it. (E) Size-exclusion chromatography of MBP-fused hMyoc-OLF and dMyoc-OLF indicated a significant aggregated fraction in hMyoc-OLF, as previously described (Burns et al., 2010), and a minor aggregated fraction in dMyoc-OLF. (Inset) SDS-PAGE analysis of dMyoc-OLF. Lane 1, molecular weight standards (kDa); lanes 2–6, size exclusion fractions as labeled in chromatogram. See also Figures S2 and S3 and Tables S2, S3, and S6.