Expression and purification of fused kinase from insect cells for in vitro kinase assay.
Expression and purification of fused kinase from insect cells for in vitro kinase assay.
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DOI：
10.1016/j.xpro.2021.100376
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发表时间：
2021-03-19
期刊：
影响因子：
--
通讯作者：
Jiang J
中科院分区：
文献类型：
--
作者：
Han Y;Jiang J
融合蛋白(FU)是Hedgehog信号的关键转导分子，但由于难以获得活性的FU用于体外检测，其相关底物尚不清楚。根据体内FU激活的机制，我们设计了一种具有结构性活性的FU，并利用杆状病毒系统在Sf9细胞中表达了它。以重组GST融合蛋白为底物，对该蛋白进行亲和纯化，并将其应用于体外蛋白激酶活性测定，以确定FU特异性的磷酸化位点。有关本议定书的使用和执行的完整细节，请参阅。从昆虫细胞中分离纯化具有活性的FU用于体外蛋白激酶活性检测用FU引发的磷酸化可以用FLAG M2亲和琼脂糖洗脱高纯度蛋白，通过pIMAGO-生物素试剂盒或特异性抗体检测3X FLAG多肽敏感的磷酸化蛋白。融合蛋白(FU)是Hedgehog信号转导的关键转导因子，但由于难以获得活性FU用于体外蛋白激酶活性检测，其相关底物一直不清楚。根据体内FU激活的机制，我们设计了一种具有结构性活性的FU，并利用杆状病毒系统在Sf9细胞中表达了它。以重组GST融合蛋白为底物，对该蛋白进行亲和纯化，并将其应用于体外蛋白激酶活性测定，以确定FU特异性的磷酸化位点。
The Fused (Fu) kinase is a key transducer of Hedgehog signaling, but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites. For complete details on the use and execution of this protocol, please refer to. Purification of constitutively active Fu from insect cells for in vitro kinase assay Priming phosphorylation by Fu can allow secondary in vitro kinase assay High-purity protein elution with Flag M2 affinity agarose and 3X Flag peptide Sensitive phospho-protein detection via pIMAGO-biotin kit or specific antibodies The Fused (Fu) kinase is a key transducer of Hedgehog signaling but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites.
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