用胆绿素和4-羟基肉桂酸对PYP-phytochrome进行纯化与重组。
Purification and reconstitution of PYP-phytochrome with biliverdin and 4-hydroxycinnamic acid.
作者信息
Chung Young-Ho, Masuda Shinji, Bauer Carl E
机构信息
Department of Biology, Indiana University, Bloomington, Indiana, USA.
出版信息
Methods Enzymol. 2007;422:184-9. doi: 10.1016/S0076-6879(06)22009-3.
PYP-phytochrome (Ppr) is a unique photoreceptor that contains a blue light-absorbing photoactive yellow protein (PYP) domain, a red light-absorbing phytochrome domain, and a histidine kinase domain. This chapter describes overexpression of Ppr in a strain of Escherichia coli that allows covalent attachment of substoichiometric amounts of biliverdin in vivo. Ppr is then fully reconstituted with biliverdin, followed by attachment of 4-hydroxycinnamic acid (p-coumaric acid), in vitro. Holo-Ppr with both chromophores is then isolated via an affinity tag and quantified for chromophore attachment by analysis of the absorption spectrum for biliverdin and 4-hydroxycinnamic acid. We also provide conditions for measuring autophosphorylation of Ppr.
PYP-植物色素（Ppr）是一种独特的光感受器，它包含一个吸收蓝光的光活性黄色蛋白（PYP）结构域、一个吸收红光的植物色素结构域和一个组氨酸激酶结构域。本章描述了Ppr在大肠杆菌菌株中的过表达，该菌株允许体内亚化学计量的胆绿素进行共价连接。然后用胆绿素对Ppr进行完全重组，接着在体外连接4-羟基肉桂酸（对香豆酸）。然后通过亲和标签分离出带有两种发色团的全Ppr，并通过分析胆绿素和4-羟基肉桂酸的吸收光谱来定量发色团的连接。我们还提供了测量Ppr自磷酸化的条件。
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