High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.
High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.
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DOI：
10.1038/srep04242
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发表时间：
2014-02-28
影响因子：
4.6
通讯作者：
Kuroda S
中科院分区：
文献类型：
--
作者：
Yoshimoto N;Tatematsu K;Iijima M;Niimi T;Maturana AD;Fujii I;Kondo A;Tanizawa K;Kuroda S
涉及单个哺乳动物跨膜受体的信号通路的重建尚未在酵母细胞中完成。在这项研究中，完整的表皮生长因子受体（EGFR）和细胞壁锚定形式的表皮生长因子共表达在酵母细胞表面，这导致表皮生长因子依赖性自分泌方式的表皮生长因子受体的自磷酸化。在从EGF改变为构象约束肽文库后，用抗磷酸化EGFR抗体荧光标记细胞。对每个细胞进行自动化单细胞分析和分离系统，该系统分析每个细胞的荧光强度并自动检索具有最高荧光的每个细胞。在~3.2 × 106肽库中，我们筛选到6个具有EGFR激动活性的新肽段。表达哺乳动物受体的酵母细胞、细胞壁锚定肽库和自动化单细胞分析和分离系统的组合可能有助于从头药物筛选的合理方法。
Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 106 peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.
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影响因子：
56.9
作者：
FINCH, EA;TURNER, TJ;GOLDIN, SM
通讯作者：
GOLDIN, SM
影响因子：
2.7
作者：
El-Haggar, Radwan;Kamikawa, Ken;Fujii, Ikuo
通讯作者：
Fujii, Ikuo
影响因子：
11.4
作者：
Lemmon, MA;Bu, ZM;Schlessinger, J
通讯作者：
Schlessinger, J
DOI：
10.1073/pnas.80.23.7080
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
EMR, SD;SCHEKMAN, R;THORNER, J
通讯作者：
THORNER, J
DOI：
10.1073/pnas.122236099
发表时间：
2002-06-11
影响因子：
11.1
作者：
Costa-Pereira, AP;Tininini, S;Poli, V
通讯作者：
Poli, V