Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

Eun Joong Oh; Rongming Liu; Liya Liang; Emily F. Freed; Carrie A. Eckert; Ryan T. Gill

doi:10.1021/acssynbio.0c00159

Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

Eun Joong Oh, Rongming Liu, Liya Liang, Emily F. Freed, Carrie A. Eckert, Ryan T. Gill

Research output: Contribution to journal › Article › peer-review

10 Scopus citations

Abstract

Advances in high-throughput synthetic biology technologies based on the CRISPR/Cas9 system have enabled a comprehensive assessment of mutations conferring desired phenotypes, as well as a better understanding of genotype-phenotype correlations in protein engineering. Engineering antibodies to enhance properties such as binding affinity and stability plays an essential role in therapeutic applications. Here we report a method, multiplex navigation of antibody structure (MINAS), that combines a CRISPR/Cas9-based trackable editing method and fluorescent-activated cell sorting (FACS) of yeast-displayed libraries. We designed mutations in all of the complementarity-determining and framework regions of a well-characterized scFv antibody and mapped the contribution of these regions to enhanced properties. We identified specific mutants that showed higher binding affinities up to 100-fold compared to the wild-type. This study expands the applicability of CRISPR/Cas9-based trackable protein engineering by combining it with a surface display platform.

Original language	English
Pages (from-to)	2197-2202
Number of pages	6
Journal	ACS Synthetic Biology
Volume	9
Issue number	8
DOIs	https://doi.org/10.1021/acssynbio.0c00159
State	Published - Aug 21 2020
Externally published	Yes

Funding

We thank Karen Helm (Cancer Center Support Grant (P30CA046934) and the Skin Diseases Research Cores Grant (P30AR057212)) for assistance and support with FACS runs performed in this study. This work was supported by the US Department of Energy (Grant DE-SC0018368) and Inscripta, Inc.

Keywords

CRISPR/Cas9
Saccharomyces cerevisiae
antibody engineering
fluorescent-activated cell sorting
yeast display

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10.1021/acssynbio.0c00159

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title = "Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform",

abstract = "Advances in high-throughput synthetic biology technologies based on the CRISPR/Cas9 system have enabled a comprehensive assessment of mutations conferring desired phenotypes, as well as a better understanding of genotype-phenotype correlations in protein engineering. Engineering antibodies to enhance properties such as binding affinity and stability plays an essential role in therapeutic applications. Here we report a method, multiplex navigation of antibody structure (MINAS), that combines a CRISPR/Cas9-based trackable editing method and fluorescent-activated cell sorting (FACS) of yeast-displayed libraries. We designed mutations in all of the complementarity-determining and framework regions of a well-characterized scFv antibody and mapped the contribution of these regions to enhanced properties. We identified specific mutants that showed higher binding affinities up to 100-fold compared to the wild-type. This study expands the applicability of CRISPR/Cas9-based trackable protein engineering by combining it with a surface display platform.",

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AU - Eckert, Carrie A.

AU - Gill, Ryan T.

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Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

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Keywords

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