A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets

Abstract

Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10 °C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein–protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications.

Virological characteristics of the novel SARS-CoV-2 Omicron variants including BA.2.12.1, BA.4 and BA.5

“…These results suggest that the mutations detected in BA.2-related Omicron variants do not affect TMPRSS2 usage. Yeast surface display assay using SARS-CoV-2 S RBD and soluble human ACE2 (Dejnirattisai et al ., 2022; Kimura et al ., 2022a; Kimura et al, 2022b; Motozono et al ., 2021; Yamasoba et al ., 2022a; Zahradnik et al, 2021a) showed that the K D value of BA.2 S RBD bearing L452R mutation is significantly lower than that of original BA.2 S RBD ( Figure 3B ), suggesting that the L452R mutation increases the binding affinity of BA.2 S RBD to human ACE2. On the other hand, the binding affinity of BA.4/5 S RBD to human ACE2 was comparable to that of BA.2 S RBD ( Figure 3B ).…”

“…Yeast surface display ( Figure 3B ) was performed as previously described as previously described (Dejnirattisai et al ., 2022; Kimura et al ., 2022a; Kimura et al ., 2022b; Motozono et al ., 2021; Yamasoba et al ., 2022a; Zahradnik et al ., 2021a). Briefly, yeast codon-optimized SARS-CoV-2_RBD-Omicron-BA.2 was obtained from Twist Biosciences and the mutant RBDs were PCR amplified by KAPA HiFi HotStart ReadyMix kit (Roche, Cat# KK2601) and assembled in vivo by yeast [ Saccharomyces cerevisiae strain EBY100 (ATCC, MYA-4941)] homologous recombination with pJYDC1 plasmid (Addgene, Cat# 162458) as previously described (Dejnirattisai et al ., 2022; Kimura et al ., 2022a; Kimura et al ., 2022b; Motozono et al ., 2021; Yamasoba et al ., 2022a; Zahradnik et al ., 2021a). Primers used are listed in Table S6 .…”

Virological characteristics of SARS-CoV-2 BA.2 variant

“…Yeast surface display ( Extended Data Fig. 6b ) was performed as previously described as previously described 22, 24, 46 . Briefly, the peptidase domain of human ACE2 (residues 18-740) was expressed in Expi293 cells and purified by a 5-ml HisTrap Fast Flow column (Cytiva, Cat# 17-5255-01) and Superdex 200 16/600 (Cytiva, Cat# 28-9893-35) using an ÄKTA pure chromatography system (Cytiva), and the purified soluble ACE2 was labelled with CF640 (Biotium, Cat# 92108).…”

Genetically engineered MRI-trackable extracellular vesicles as SARS-CoV-2 mimetics for mapping ACE2 bindingin vivo

“…Protein production, purifications, and labeling procedures: The designed ALFA-tag binding nanobody (DnbALFA) and its mNeonGreen fusion were expressed by using expression plasmid pET28bdSUMO 62 and E.coli BL21(DE3) cells as described previously 60 . Briefly, 200 ml of 2YT medium (16 g tryptone, 10 g yeast extract and 5 g NaCl, pH 7) was inoculated (1%), grown to the OD600 = 0.6 (37°C), and the expression was initiated by the addition of IPTG to a final concentration of 0.5 mM.…”

“…The pAGDisplay vector backbone was assembled by combining a pET28b fragment bearing KanR and origin of replication, a pLVX vector fragment bearing WPRE, PuroR, and IRES sequences, and a pDisplay CMV promoter with a PDGFRβ expression cassette by a restriction-free three-component assembly 58 . In the subsequent restriction-free cloning step, the PuroR was fused with eUnaG2 fluorescent protein at the C-terminus 59,60 . The full-length pAGDisplay was sequenced to verify its correct assembly.…”