Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates.
Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates.
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DOI：
10.1021/ja412934t
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发表时间：
2014-02-26
影响因子：
15
通讯作者：
Liu DR
中科院分区：
文献类型：
--
作者：
McGregor LM;Jain T;Liu DR
我们描述了使用未纯化蛋白 (IDUP) 相互作用测定的开发和验证，这是一种选择性扩增 DNA 序列的方法，从细胞裂解物中的 DNA 连接小分子和未纯化蛋白靶标的混合物中识别配体+靶标对。通过在细胞裂解物中进行操作，IDUP 保留了天然的翻译后修饰以及与内源结合伴侣的相互作用，从而能够研究难以纯化的靶标，并与需要纯化蛋白质的方法相比，增加了检测到的相互作用的潜在生物学相关性。在 IDUP 中，目标蛋白与 DNA 寡核苷酸标签相关，或者使用 DNA 连接抗体非共价连接，或者使用 SNAP 标签共价连接。配体-靶标结合促进自启动发夹的杂交，该发夹由 DNA 聚合酶延伸以产生包含识别靶标及其配体的序列的 DNA 链。这些编码配体+靶标对的序列通过 PCR 选择性扩增，并通过高通量 DNA 测序揭示。 IDUP 可以对细胞裂解物中亲和力调节接头蛋白的作用做出反应，而使用纯化的蛋白质靶标的配体筛选或选择方法中将不存在这种作用。这种能力通过在过表达 FRB 和 FKBP 的样品中编码 FRB+雷帕霉素或 FKBP+雷帕霉素的 DNA 序列扩增 100 倍来例证（FRB·雷帕霉素+FKBP，Kd ≈ 100 fM；FKBP·雷帕霉素+FRB，Kd = 12 nM）。相比之下，在单独过表达 FRB 或 FKBP 的样品中，这些序列的扩增效率低 10 倍（雷帕霉素+FKBP，Kd ≈ 0.2 nM；雷帕霉素+FRB，Kd = 26 μM）。最后，IDUP 用于处理单个样品中组合的 DNA 连接小分子模型库和表达 SNAP-靶融合物的细胞裂解物模型库。在这个文库×文库实验中，IDUP 导致在 67,858 个可能的组合中富集了与 5 个已知配体+靶标对相对应的序列，其结合亲和力范围为 Kd = 0.2 nM 至 3.2 μM，并且没有假阳性信号富集到与任何真正的配体+靶标对相同的程度。
We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand–target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding affinity from Kd = 0.2 nM to 3.2 μM out of 67,858 possible combinations, with no false positive signals enriched to the same extent as that of any of the bona fide ligand+target pairs.
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