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. 2019 Nov 26;7:362. doi: 10.3389/fbioe.2019.00362

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Copyright © 2019 Mei, Li, Wang, Lee, Iverson, Zhang, Ge and Yi.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Fab yeast surface display using a divergent promoter, ER-targeting signals, and Aga2 fusion. (A) pESD-Fab. Expression cassettes for light (V_L-C_L) and heavy (V_H-C_H1) chains were located downstream of GAL1-GAL10 promoter and endoplasmic reticulum (ER)-targeting signals. Light chain was fused with a HA tag, and heavy chain was fused with a FLAG tag to the N-terminus of Aga2. (B) Validation of heavy chain display (left panels), light chain display (middle panels), and Fab assembly (right panels) on yeast cell surface by flow cytometry. Expression was induced by 20 g/L galactose and cells were incubated with 0.1 μM anti-FLAG-iFluor647 and/or 0.1 μM anti-HA-FITC antibodies. Fab display of Adalimumab (D2E7) and three variants were tested. Percentages of cells displaying assembled Fabs were shown as Q2 (FLAG⁺/HA⁺ double positive). MFI, mean fluorescence intensity.