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Magnetic Bead-Immobilized Mammalian Cells Are Effective Targets to Enrich Ligand-Displaying Yeast.
ACS Combinatorial Science ( IF 3.903 ) Pub Date : 2020-04-27 , DOI: 10.1021/acscombsci.0c00036
Patrick S Lown 1 , Benjamin J Hackel 1
Affiliation  

Yeast surface display empowers selection of protein binding ligands, typically using recombinant soluble antigens. However, ectodomain fragments of transmembrane targets may fail to recapitulate their true, membrane-bound form. Direct selections against adhered mammalian cells empower enrichment of genuine binders yet benefit from high target expression, robustly adherent mammalian cells, and nanomolar affinity ligands. This study evaluates a modified format with mammalian cells immobilized to magnetic beads; yeast-displayed fibronectin domain and affibody ligands of known affinities and cells with expression ranges of epidermal growth factor receptor (EGFR) and CD276 elucidate important parameters to ligand enrichment and yield in cell suspension panning with comparison to adherent panning. Cell suspension panning is hindered by significant background of nondisplaying yeast but exhibits yield advantages in model EGFR systems for a high affinity (KD = 2 nM) binder on cells with both high (106 per cell) target expression (9.6 ± 0.6% vs 3.2 ± 0.4%, p < 0.0001) and mid (105) target expression (2.3 ± 0.5% vs 0.41 ± 0.09%, p = 0.0008), as well as for a low affinity (KD > 600 nM) binder on high target expression cells (2.0 ± 0.5% vs 0.017 ± 0.005%; p = 0.001). Significant enrichment was observed for all EGFR systems except the low-affinity, high expression system. The CD276 system failed to provide significant enrichment, indicating that this technique may not be suitable for all targets. Collectively, this study highlights new approaches that yield successful enrichment of yeast-displayed ligands via panning on immobilized mammalian cells.

中文翻译:

磁珠固定的哺乳动物细胞是丰富显示配体的酵母的有效靶标。

酵母表面展示通常可以使用重组可溶性抗原来选择蛋白质结合配体。但是,跨膜靶标的胞外域片段可能无法概括其真实的,膜结合的形式。针对粘附的哺乳动物细胞的直接选择可以增强真正的结合剂,但得益于高靶标表达,牢固粘附的哺乳动物细胞和纳摩尔亲和配体。这项研究评估了将哺乳动物细胞固定在磁珠上的改良形式。酵母展示的已知亲和力的纤连蛋白结构域和亲和体配体以及具有表皮生长因子受体(EGFR)和CD276的表达范围的细胞阐明了细胞悬液淘选中与配体淘选相比配体富集和产量的重要参数。细胞悬浮淘选受到不展示酵母的显着背景的阻碍,但在模型EGFR系统中显示了对具有高(每细胞106个)目标表达(9.6±0.6%vs 3.2±)的细胞具有高亲和力(KD = 2 nM)结合剂的产量优势。 0.4%,p <0.0001)和中位(105)靶标表达(2.3±0.5%vs 0.41±0.09%,p = 0.0008)以及在高靶标表达细胞上的低亲和力(KD> 600 nM)结合剂( 2.0±0.5%和0.017±0.005%; p = 0.001)。除低亲和力,高表达系统外,所有EGFR系统均观察到显着富集。CD276系统未能提供足够的富集,表明该技术可能不适用于所有靶标。总的来说,
更新日期:2020-04-13
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