Expression of green fluorescent protein fused to magnetosome proteins in microaerophilic magnetotactic bacteria
Expression of green fluorescent protein fused to magnetosome proteins in microaerophilic magnetotactic bacteria
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DOI：
10.1128/aem.00231-08
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发表时间：
2008-08-01
影响因子：
4.4
通讯作者：
Schueler, Dirk
中科院分区：
文献类型：
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作者：
Lang, Claus;Schueler, Dirk
趋磁细菌的磁小体是原核生物的细胞器，由磷脂双层包围的磁铁矿晶体组成，磷脂双层包含一组具有各种功能的蛋白质。由于其独特的磁性和结晶性质，磁小体颗粒在许多应用中可能用作磁性纳米颗粒，这在许多情况下需要将功能部分偶联到磁小体膜。在这项工作中，我们研究了使用绿色荧光蛋白（GFP）作为一个报告的磁小体定位和表达的融合蛋白在微需氧的Magnetocellum gryphiswaldense细胞计数，荧光显微镜，和生化分析。虽然高荧光和磁铁矿合成的最佳条件是相互排斥的，我们建立了氧气限制的生长条件，支持增长，磁铁矿生物矿化，并在合理的速率GFP荧光团的形成。在这些优化的条件下，我们研究了亚细胞定位和表达的GFP标记的磁小体蛋白MamC，MamF，和MamG的荧光显微镜和免疫印迹。虽然所有融合体都特异性地定位于磁小体膜，但MamC-GFP显示出最强的表达和荧光。从细胞中纯化的MamC-GFP标记的磁小体显示出强烈的荧光，其对去污剂敏感，但在宽范围的温度和盐浓度下稳定。总之，我们的数据表明，使用GFP作为报告的磁铁矿形成条件下的蛋白质定位和MamC作为锚的磁小体特异性显示异源基因融合的效用。
The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by How cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.