Evaluation of the yeast surface display system for screening of functional nanobodies

Mitsuyoshi Ueda,Wataru Aoki,Kaho Kajiwara

doi:10.1186/s13568-020-00983-y

Abstract

Yeast surface display is a powerful technology used to isolate and engineer proteins to improve their activity, specificity, and stability. In this method, gene expression is regulated by promoters, and secretion efficiency is affected by secretion signals. Furthermore, both the accessibility and activity of the displayed proteins are affected by the length of anchor proteins. The ideal promoter, secretion signal, and anchor protein combination depend on the proteins of interest. In this study, we optimized a yeast surface display suitable for nanobody evaluation. We designed five display systems that used different combinations of promoters, secretion signals, and anchor proteins. Anti-hen egg-white lysozyme nanobody was used as the model nanobody. The amount of nanobodies displayed on yeast cells, the number of antigens bound to the displayed nanobodies, and the display efficiency were quantified. Overall, we improved the yeast display system for nanobody engineering and proposed its optimization.

Highlights

Camelid single-domain antibody fragments (“nanobodies”) are being increasingly utilized in various applications (Salvador et al 2019)
A HA-tag and anchor protein were fused to the C-terminus of the nanobody sequence because the complementary-determining regions of nanobodies exist at their N-terminus
The fluorescence intensities of Alexa Fluor® 488 (AF488) and Alexa Fluor® 647 (AF647) were higher under the GAP promoter than under the GAL1 promoter (Fig. 6a, b [left and middle panels]). These data indicate that the GAP promoter was stronger than the GAL1 promoter; the ratio of yeast cells in the Q2 region was higher under the GAL1 promoter than under the GAP promoter (Fig. 6a, b [right panels])

Summary

Introduction

Camelid single-domain antibody fragments (“nanobodies”) are being increasingly utilized in various applications (Salvador et al 2019). Their most notable advantage is their small size; the molecular weight of one nanobody is approximately 15 kDa, i.e., one-tenth that of conventional IgG (Hamers-Casterman et al 1993). Yeast surface display is a potent screening platform used for protein engineering (Boder and Wittrup 1997; McMahon et al 2018; Ueda 2019). In the yeast surface display system, a gene encoding a protein of interest with a secretion signal peptide is fused with a gene encoding an anchor protein This fusion gene is introduced into yeast cells and expressed under the control of any promoter. Yeast cells are compatible with flow cytometry, allowing for quantitative screening (Ueda 2019)

Methods

Results

Conclusion