双功能酵母展示与分泌系统的构建与验证

Generation and verification of dual functional yeast display/secretion system

酵母展示技术被广泛应用于抗体药物候选分子的发现与优化中,但传统酵母展示技术只能将抗体展示于表面,无法同时将抗体分泌至培养基的上清液进行亲和力测定。为克服此难点,将含Knob突变的抗体可结晶片断(Fc)展示于酵母表面作为“诱饵”,通过“Knob into Hole”的方式结合含Hole突变的免疫球蛋白(IgG)半分子,达到展示抗体的作用;同时,含Hole突变的IgG半分子自身可形成同源二聚体,形成IgG完整分子分泌至培养基中,实现酵母抗体展示与分泌的双重功能。结果发现:使用含Knob突变的重链恒定区3作为“诱饵”,展示抗体的效果最佳;双功能酵母展示与分泌系统经模拟文库验证,可高效筛选出占比极低的含高亲和力抗体序列的菌株;筛选出的菌株可将抗体同时分泌至培养基上清,直接用于亲和力测定。本研究结果充分说明,双功能酵母展示与分泌系统搭建成功并可用于抗体的亲和力成熟中,从而显著提高工作效率、缩短抗体亲和力成熟的周期。

Yeast surface display is widely used in antibody discovery and optimization.However,traditional yeast display system could only display the antibody on the cell surface without the secretion of antibody to the cell culture supernatant for direct affinity measurement.To solve this problem,we generated the dual functional yeast display/secretion system that displayed antibody crystallizable fragments(Fc)with Knob modified as“bait”to capture half molecular immunoglobulin(IgG)with Hole mutation,and secreted fully IgG assembled by Hole-Hole homodimer to the culture supernatant.Results showed that the heavy chain constant region 3 of immunoglobulin with Knob mutation was the best“bait”for antibody display.The dual functional yeast display/secretion system could not only efficiently identify the strains displayed high affinity antibodies from spiking library but also secrete the antibodies to the supernatant for direct affinity measurement.This study proves that the dual functional yeast display/secretion system was successfully used for antibody affinity maturation,resulting in marked acceleration of the process.