An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea.
An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea.
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DOI：
10.1186/1472-6750-13-112
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发表时间：
2013-12-21
影响因子：
3.5
通讯作者：
Smith B
中科院分区：
文献类型：
--
作者：
DasSarma S;Karan R;DasSarma P;Barnes S;Ekulona F;Smith B
气体囊泡是中空的，漂浮的细胞器，由薄而非常稳定的蛋白质膜包围。它们由嗜盐古菌Halobacterium sp. NRC-1中的一组gvp基因编码。使用含有整个gvp基因簇的表达载体，通过在gvpC基因与来自细菌和病毒病原体的编码序列之间构建基因融合物，已经成功地将气泡纳米颗粒（GVNPs）生物工程化用于抗原展示。为了改进和简化用于GVNPs生物工程的遗传系统，我们首先构建了仅缺失gvpC基因的盐杆菌属NRC-1菌株。删除的菌株含有更小，更纺锤形的纳米粒子可观察到的透射电子显微镜，确认形状决定的作用GvpC在气泡生物发生。接下来，我们构建了含有N-末端编码部分或完整gvpC基因的表达质粒。将表达质粒导入盐杆菌NRC-1 ΔgvpC菌株后，通过Western印迹分析将GvpC蛋白和变体定位于GVNPs，并通过电子显微镜确定其对增加纳米颗粒大小和形状的影响。最后，将编码Gaussia princeps荧光素酶的合成基因与表达质粒上的gvpC基因片段融合，产生与来自盐杆菌的漂浮纳米颗粒结合的酶活性GvpC-荧光素酶融合蛋白。从质粒构建体表达的GvpC蛋白及其N-末端片段补充了盐杆菌属NRC-1 ΔgvpC菌株，并与漂浮的GVNPs结合。来自Gaussia princeps的荧光素酶报告基因与表达质粒中的gvpC基因衍生物的融合产生了结合有酶活性荧光素酶的GVNPs。这些结果建立了一个显着改善的遗传系统，用于显示外源蛋白的盐杆菌气泡和扩展这些新的纳米粒子的生物工程潜力，催化活性酶。
Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1. Using an expression vector containing the entire gvp gene cluster, gas vesicle nanoparticles (GVNPs) have been successfully bioengineered for antigen display by constructing gene fusions between the gvpC gene and coding sequences from bacterial and viral pathogens. To improve and streamline the genetic system for bioengineering of GVNPs, we first constructed a strain of Halobacterium sp. NRC-1 deleted solely for the gvpC gene. The deleted strain contained smaller, more spindle-shaped nanoparticles observable by transmission electron microscopy, confirming a shape-determining role for GvpC in gas vesicle biogenesis. Next, we constructed expression plasmids containing N-terminal coding portions or the complete gvpC gene. After introducing the expression plasmids into the Halobacterium sp. NRC-1 ΔgvpC strain, GvpC protein and variants were localized to the GVNPs by Western blotting analysis and their effects on increasing the size and shape of nanoparticles established by electron microscopy. Finally, a synthetic gene coding for Gaussia princeps luciferase was fused to the gvpC gene fragments on expression plasmids, resulting in an enzymatically active GvpC-luciferase fusion protein bound to the buoyant nanoparticles from Halobacterium. GvpC protein and its N-terminal fragments expressed from plasmid constructs complemented a Halobacterium sp. NRC-1 ΔgvpC strain and bound to buoyant GVNPs. Fusion of the luciferase reporter gene from Gaussia princeps to the gvpC gene derivatives in expression plasmids produced GVNPs with enzymatically active luciferase bound. These results establish a significantly improved genetic system for displaying foreign proteins on Halobacterium gas vesicles and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.
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影响因子：
3.5
作者：
Karan, Ram;Capes, Melinda D.;DasSarma, Shiladitya
通讯作者：
DasSarma, Shiladitya
影响因子：
4.4
作者：
Chu, Lichieh Julie;Chen, Mengchieh Claire;Ng, Wailap Victor
通讯作者：
Ng, Wailap Victor
影响因子：
5.5
作者：
Childs, Tawanna S.;Webley, Wilmore C.
通讯作者：
Webley, Wilmore C.
影响因子：
3.2
作者：
DASSARMA, S;ARORA, P;YIN, LRS
通讯作者：
YIN, LRS
DOI：
10.1186/1746-1448-3-6
发表时间：
2007-07-25
期刊：
Saline systems
影响因子：
--
作者：
Coker JA;DasSarma P;Kumar J;Müller JA;DasSarma S
通讯作者：
DasSarma S