Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein
Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein
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DOI：
10.1007/s12275-008-0188-x
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发表时间：
2009-04-01
影响因子：
3
通讯作者：
Fa-aroonsawat, Sirirat
中科院分区：
文献类型：
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作者：
Chungjatupornchai, Wipa;Fa-aroonsawat, Sirirat
由于高度分化的膜系统的存在，蛋白质向蓝藻细胞包膜的易位变得复杂。为了使用来自丁香假单胞菌 KCTC 1832 的截短冰核蛋白 (InpNC) 研究蓝藻聚球藻 PCC 7942 中的蛋白质易位，将绿色荧光蛋白 (GFP) 框内融合到 InpNC 的羧基末端。发现 GFP 的荧光几乎完全作为细胞外部区域的光晕，如共焦激光扫描显微镜所证明的，该区域似乎对应于周质，然而，GFP 并未显示在最外层的细胞表面上。 Western blotting分析显示InpNC-GFP融合蛋白部分降解。 InpNC 的 N 端结构域可能容易受到蛋白酶攻击；与 GFP 缀合的剩余 C 端结构域失去了直接跨外膜易位并充当表面展示基序的能力。具有周质 GFP 的细胞的荧光强度比具有细胞质 GFP 的细胞低约 6 倍。活性 GFP 成功易位至周质可能为研究蓝藻周质物质以非侵入性方式响应环境变化的特性提供了一种潜在的手段。
The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner.