摘要
The mechanism of action of H,K-ATPase is still only partly understood. Site-directed mutagenesis and chimere production might give useful tools to enlarge our insight in structure-function relationship of this enzyme. Functional expression in vitro is a prerequisite for such studies. The baculovirus expression system makes advantage of the high level expression of certain viral proteins not essential for viral replication in a cell line from the fall armyworm Spodoptera frugiperda (Sf9 cells) [Vlak and Keus,1990]. Two such proteins are polyhedrin and p10 protein. Both proteins can reach expression levels of more than 30% in Sf9 cells, dependent on the stage of infection. Replacing polyhedrin or p10 coding sequences by homologous recombination between wild-type viral DNA and a transfer vector DNA has led to the production of recombinant viruses expressing large amounts of recombinant proteins. Many complex membrane proteins as well as cytosolic proteins have successfully been expressed using this system [Parker et al.,1991; Janssen et al.,1991; Fafournoux et al.,1991; Li et al.,1992; Smith et al.,1992; De Tomaso et al.,1993]. In order to produce multiple subunit proteins, simultaneous expression of two or more proteins has been made possible by using either one transfer vector containing two or more promoters [Belyaev and Roy,1993] or by co-infection of Sf9 cells using a mixture of two or more recombinant baculoviruses. We therefore decided to study the in vitro synthesis of H,K-ATPase subunits using the baculovirus expression system.
摘要译文
H，K-ATP酶的作用机制仍只有部分理解。定点诱变和嵌合体生产可能会提供有用的工具来扩大我们对这种酶的结构 - 功能关系的洞察力。体外功能表达是此类研究的先决条件。系统利用了来自秋粘虫Spodoptera frugiperda（Sf9细胞）的细胞系中对于病毒复制不是必需的某些病毒蛋白的高水平表达[Vlak和Keus，1990]。两种这样的蛋白质是多角体蛋白和p10蛋白质。这两种蛋白在Sf9细胞中的表达水平可以达到30％以上，这取决于感染的阶段。通过野生型病毒DNA和转移载体DNA之间的同源重组产生p10编码序列导致产生表达大量重组蛋白的重组病毒。许多复杂的膜蛋白以及细胞溶质蛋白已经使用该系统成功表达[Parker等，1991; Janssen等，1991; Fafournoux等，1991;李等人，1992;Smith等人，1992; De Tomaso等，1993]。为了产生多个亚基蛋白质，通过使用含有两个或更多个启动子的一种转移载体[Belyaev和Roy，1993]或通过使用两种或更多种重组杆状病毒的混合物共同感染Sf9细胞。因此我们决定研究H的体外合成，K-ATPase亚基使用杆状病毒表达系统。
Corné H. W. Klaassen1;Tom J. F. Van Uem1;Mariëlle P. De Moel1;Godelieve L. J. De Caluwé1;Herman G. P. Swarts1;Jan Joep H. H. M. De Pont1. Functional Expression of Gastric H,K-ATPase in Sf9 Cells using the Baculovirus Expression System. Molecular and Cellular Mechanisms of H+ Transport[M].DE: Springer, 1994: 19-26