Paulk Alexandra M, Williams Rory L, Liu Chang C
bioRxiv. 2024 May 24:2024.05.24.595666. doi: 10.1101/2024.05.24.595666.
We recently developed 'autonomous hypermutation yeast surface display' (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence activated cell sorting (FACS). AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 hours of induction. Here, we report an updated version of the AHEAD platform that utilizes a synthetic β-estradiol induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system as well as traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.
我们最近开发了“自主高突变酵母表面展示”（AHEAD）技术，该技术能够在酵母中快速产生强效且特异性的抗体。AHEAD将酵母表面展示与易错正交DNA复制系统（OrthoRep）相结合，以持续快速地突变表面展示的抗体，从而通过重复的细胞生长和荧光激活细胞分选（FACS）轮次富集更强结合变体。AHEAD目前利用标准的半乳糖诱导系统来驱动抗体在酵母表面的选择性展示。然而，达到最大展示水平可能需要长达48小时的诱导。在此，我们报告了AHEAD平台的一个更新版本，该版本利用合成的β-雌二醇诱导基因表达系统来调节抗体的表面展示，并发现无论是我们的AHEAD系统还是来自不发生高突变的核质粒的传统酵母表面展示，诱导在实现表面展示方面都明显更快。更新后的AHEAD平台在重复的进化轮次中完全发挥功能，以驱动抗体的快速进化。
