A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.
A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.
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DOI：
10.1093/protein/gzr021
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发表时间：
2011-07
期刊：
影响因子：
--
通讯作者：
Waldo GS
中科院分区：
文献类型：
--
作者：
Lockard MA;Listwan P;Pedelacq JD;Cabantous S;Nguyen HB;Terwilliger TC;Waldo GS
我们描述了一个体外菌落筛选，以鉴定大肠杆菌表达可溶性蛋白和稳定，组装多蛋白复合物。带有n端6His标签和c端绿色荧光蛋白（GFP） S11标签的蛋白质通过与共表达的GFP 1-10片段互补在细胞中进行荧光标记。部分菌落裂解后，荧光可溶性蛋白或复合物通过支撑过滤膜扩散，并捕获在固定在琼脂糖中的Talon®树脂金属亲和珠上。荧光菌落的图像传达了总表达，结合在珠上的荧光水平表明有多少蛋白质是可溶的。在选择克隆时，这两个信息可以一起使用。化验后，菌落可以采摘和繁殖，不需要制作复制板。我们使用该方法筛选人类p85蛋白的DNA片段文库，并优先获得表达全长“断点簇区域同源性”和NSH2结构域的克隆。该试验还区分了表达稳定的多蛋白复合物的克隆和那些由于缺少亚基而不稳定的克隆。在以缺失N亚基的YheML复合物为主的文库中，很容易鉴定出表达稳定、完整的异三聚体大肠杆菌YheNML复合物的克隆。
We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.
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影响因子：
8
作者：
Armstrong, N;De Lencastre, A;Gouaux, E
通讯作者：
Gouaux, E
影响因子：
8
作者：
Goda, N;Tenno, T;Shirakawa, M
通讯作者：
Shirakawa, M
影响因子：
46.9
作者：
Cabantous, S;Terwilliger, TC;Waldo, GS
通讯作者：
Waldo, GS
DOI：
10.1107/s174430910901207x
发表时间：
2009-05-01
影响因子：
0.9
作者：
Garcia, Brandon L.;Tzekou, Apostolia;Geisbrecht, Brian V.
通讯作者：
Geisbrecht, Brian V.
影响因子：
8
作者：
Jacobs, SA;Podell, ER;Cech, TR
通讯作者：
Cech, TR