Rapidly Inducible Yeast Surface Display for Antibody Evolution with OrthoRep.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2024-08-16 Epub Date: 2024-07-25 DOI:10.1021/acssynbio.4c00370

Alexandra M Paulk, Rory L Williams, Chang C Liu

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Abstract

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. 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 h 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 and 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.

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利用 OrthoRep 快速诱导酵母表面显示技术实现抗体进化

我们最近开发了 "自主超突变酵母表面展示"（AHEAD）技术，该技术能够在酵母中快速产生强效特异性抗体。AHEAD将酵母表面展示与易出错的正交DNA复制系统（OrthoRep）配对，持续、快速地突变表面展示的抗体，从而通过反复的细胞生长和荧光激活细胞分选，富集出更强的结合变体。目前，AHEAD 利用标准半乳糖诱导系统来驱动抗体在酵母表面的选择性显示。然而，要达到最大显示水平可能需要长达 48 小时的诱导。在这里，我们报告了 AHEAD 平台的升级版，它利用合成的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 系统还是传统的酵母表面展示，诱导的速度都明显更快。更新后的 AHEAD 平台在反复的进化过程中完全发挥作用，推动了抗体的快速进化。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.