Ntefidou Maria, Lüdtke Timo, Ahmad Margaret, Häder Donat-Peter
Friedrich-Alexander Universität, Institut für Biologie, Erlangen, Germany.
Photochem Photobiol. 2006 Nov-Dec;82(6):1601-5. doi: 10.1562/2006-04-06-RA-867.
The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACalpha (Mr 105 kDa) and two PACbeta (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of over-expression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coli. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACalpha and PACbeta were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACbeta and PACbeta, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACalpha and PACbeta antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.
单细胞绿色鞭毛虫野生型纤细裸藻（Z菌株）拥有两个光激活腺苷酸环化酶（PAC）家族的基因。相应的基因产物被发现负责增强型（而非减弱型）避光反应以及正向和负向光趋性。这些蛋白质由两个PACα（分子量105 kDa）和两个PACβ（90 kDa）亚基组成。为了产生足够量的PAC蛋白，人们尝试了几种过表达途径，包括在裸藻中的同源表达和在大肠杆菌中的异源表达。所有这些方法都受到低产量或包涵体形成的阻碍。因此，我们决定尝试在昆虫细胞系中进行异源表达。PACα和PACβ分别克隆到带有His标签的转移载体pBacPAK9中。随后，该转移载体通过杆状病毒共转染到昆虫细胞中并进行扩增。为了表达，两种重组病毒（分别含有PACα和PACβ）同时共转染到昆虫细胞中。使用PACα和PACβ抗体通过蛋白质免疫印迹法分析表达的蛋白质。发现大多数蛋白质以高产率呈可溶形式。重组PAC蛋白通过其在抗His树脂上附着的His标签进行纯化。使用cAMP酶免疫分析试剂盒在蓝光激发后对腺苷酸环化酶活性进行定量。
