来自群居仙水母的一种光蛋白——仙水母荧光蛋白的表达、纯化及特性分析
Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium.
作者信息
Inouye Satoshi, Sahara Yuiko
机构信息
Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan.
出版信息
Protein Expr Purif. 2007 Jun;53(2):384-9. doi: 10.1016/j.pep.2006.12.014. Epub 2006 Dec 24.
A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20mg from 2L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca(2+) sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca(2+). Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.
构建了一种新型的带有组氨酸标签的大肠杆菌分泌载体，并制备了大量高纯度的水母发光蛋白clytin，一种钙结合光蛋白。通过镍螯合亲和层析法纯化在大肠杆菌周质空间中表达的带有组氨酸标签的脱辅基水母发光蛋白。在二硫苏糖醇存在下，通过与腔肠素孵育，从脱辅基水母发光蛋白中再生重组水母发光蛋白，然后通过阴离子交换层析和疏水层析进行纯化。从2升培养细胞中获得的重组水母发光蛋白产量为20毫克，纯度大于95%。重组水母发光蛋白的发光特性与天然水母发光蛋白（水母素）相同。重组水母发光蛋白对Ca(2+)的敏感性低于水母发光蛋白，适合用于测量较高浓度的Ca(2+)。还使用腔肠素类似物制备了半合成水母发光蛋白，并对其初始强度、发光能力和半衰期进行了表征。
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