Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum
Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum
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DOI：
10.1023/a:1024498805771
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发表时间：
2002-10-01
影响因子：
2.7
通讯作者：
Manstein, DJ
中科院分区：
文献类型：
--
作者：
Knetsch, MLW;Tsiavaliaris, G;Manstein, DJ
我们生成并测试了一组克隆载体，其设计用于促进盘基网柄藻中蛋白质的生产、纯化和可视化。载体来源于网柱藻-E。大肠杆菌穿梭载体pDXA-3 H（6.1kb）携带Dd高拷贝质粒Ddp 2的复制起点，Ddp 2是一个高拷贝的大肠杆菌。coli质粒复制起点、act 6启动子驱动的G418抗性盒、细菌氨苄青霉素抗性基因和表达盒。新的克隆载体携带由强组成型肌动蛋白-15启动子组成的表达盒，翻译起始点，随后是多克隆位点，用于添加纯化或可视化标签的序列，以及Dictyosteomycin多聚腺苷酸化和终止信号。设计用于促进活网骨藻细胞中蛋白质可视化的载体含有绿色荧光蛋白（GFP）的青色（CFP）或黄色（YFP）变体的编码序列。产生了用于产生N和C-末端与荧光蛋白融合的载体形式。为了促进蛋白质纯化，产生用于产生谷胱甘肽-S-转移酶（GST）融合蛋白和Strep-或FLAG-亲和标记的蛋白质的载体。另外，产生了用于产生His（8）-标记的蛋白质的载体，其具有被潮霉素抗性盒替换的G418抗性盒。
We generated and tested a set of cloning vectors designed to facilitate the production, purification and visualization of proteins in Dictyostelium discoideum. The vectors are derived from the Dictyostelium-E. coli shuttle vector pDXA-3H (6.1 kb), which carries the origin of replication of the Dd high-copy-number plasmid, Ddp2, a high-copy-number E. coli plasmid origin of replication, an act6 promoter driven G418 resistance cassette, the bacterial ampicillin resistance gene and an expression cassette. The new cloning vectors carry expression cassettes consisting of the strong constitutive actin-15 promoter, a translation start followed by a multiple cloning site, sequences for the addition of purification or visualization tags, and Dictyostelium polyadenylation and termination signals. Vectors designed to facilitate protein visualization in living Dictyostelium cells contain either coding sequences for the cyan (CFP) or yellow (YFP) variants of green fluorescent protein (GFP). Versions of the vectors for the production of Nand C-terminal fusions with the fluorescent proteins were generated. To facilitate protein purification, vectors for the production of glutathione-S-transferase (GST) fusion proteins and Strep- or FLAG-afinity-tagged proteins were generated. Additionally, a vector for the production of His(8)-tagged proteins was generated, which has the G418-resistance cassette replaced by a hygromycin resistance cassette.