Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.
Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.
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扩展的酵母表面展示接头增强了直接哺乳动物细胞选择中配体的富集。
DOI：
10.1093/protein/gzab004
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Hackel,BenjaminJ
中科院分区：
文献类型：
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作者：
Lown,PatrickS;Cai,JessyJ;Ritter,SethC;Otolski,JacobJ;Wong,Ryan;Hackel,BenjaminJ
哺乳动物细胞单层上的酵母展示配体的选择受益于高靶标表达和纳摩尔亲和力，这并不总是可用的。先前的工作将酵母-蛋白质接头从40个氨基酸延伸到80个氨基酸提高了产率和富集，但假设低于最佳长度，从而促进了对延伸的氨基酸接头的评估。641个残基的连接子提供了增强的富集，其中2 nM亲和性纤连蛋白配体和105个表皮生长因子受体（EGFR）/细胞（14 ± 2 vs. 8 ± 1，P= 0.008）和>600 nM亲和性配体，106个EGFR/细胞系统（23 ± 7 vs. 0.8 ± 0.2，P = 0.004）。用310-nM亲和力抗体配体和104个CD 276/细胞也观察到增强的富集，这表明对其他支架和靶标的可推广的益处。连接体的空间建模表明，配体的改善的细胞外可及性使得能够在先前不可能的条件下观察到富集。
Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast–protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105epidermal growth factor receptors (EGFR) per cell (14 ± 2 vs. 8 ± 1,P= 0.008) and a >600-nM affinity ligand, 106EGFR per cell system (23 ± 7 vs. 0.8 ± 0.2,P= 0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.