Mei Meng, Li Junhong, Wang Shengchen, Lee Ki Baek, Iverson Brent L, Zhang Guimin, Ge Xin, Yi Li
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, China.
Department of Chemical and Environmental Engineering, University of California, Riverside, Riverside, CA, United States.
Front Bioeng Biotechnol. 2019 Nov 26;7:362. doi: 10.3389/fbioe.2019.00362. eCollection 2019.
For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:10 or 1:10 spiked libraries.
对于抗体发现和工程而言，抗原结合片段（Fabs）的酵母表面展示（YSD）以及耦合荧光激活细胞分选（FACS）可提供完整的互补位构象，并在单克隆水平进行定量分析，因此在众多应用中具有巨大潜力。本研究以抗TNFα单克隆抗体英夫利昔单抗、阿达木单抗及其变体作为模型Fabs，系统地描述了优化Fab YSD的互补方法。结果表明，通过使用不同的启动子和内质网（ER）信号肽来共表达轻链和重链 - Aga2融合蛋白，组装好的Fabs以对TNFα呈S形结合反应的方式功能性地展示在酵母细胞表面。热休克蛋白70家族分子伴侣Kar2p和/或蛋白二硫键异构酶（Pdi1p）的共表达显著提高了功能性展示的效率（定义为展示功能性Fab的细胞与展示组装好的Fab的细胞的比例）。此外，将ER保留序列（ERSs）与轻链融合也以展示量为代价提高了Fab的展示质量，并且改善程度与ERSs的强度相关，对英夫利昔单抗而言比对阿达木单抗更显著。通过从1:10或1:10加标的文库中分离出高亲和力Fab克隆，进一步证明了亲和力成熟的可行性。
