Molecular characterization of an ice nucleation protein variant (inaQ) from Pseudomonas syringae and the analysis of its transmembrane transport activity in Escherichia coli.
Molecular characterization of an ice nucleation protein variant (inaQ) from Pseudomonas syringae and the analysis of its transmembrane transport activity in Escherichia coli.
复制标题
丁香假单胞菌冰核蛋白变体 (InaQ) 的分子表征及其在大肠杆菌中的跨膜转运活性分析
DOI：
10.7150/ijbs.4524
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发表时间：
2012
影响因子：
9.2
通讯作者：
Li L
中科院分区：
文献类型：
--
作者：
Li Q;Yan Q;Chen J;He Y;Wang J;Zhang H;Yu Z;Li L
丁香假单胞菌的冰核蛋白(INP)不仅因为其对叶部坏死的致病性，而且在制雪、冷冻食品制备和表面展示系统开发等方面具有广泛的潜在应用价值，因此引起了科学研究的兴趣。然而，目前对INP转运活性的研究还很少。在本研究中，从一株丁香疫霉菌MB03株中克隆了一个新发现的INP基因变异体inaQ。然后对其结构域、信号序列以及每个结构域的亲水性或疏水性进行了表征。其推导的氨基酸序列与三个丁香疫霉INP，即Inak、InaZ和iNAV具有相似的蛋白质结构域，分别被鉴定为N-末端结构域、中心重复结构域和C-末端结构域。以绿色荧光蛋白(GFP)为融合伙伴，在大肠杆菌中诱导表达全长InaQ和各种截短突变体，分析其跨膜转运和表面结合活性。由于有两个跨膜片段和一个微弱的分泌信号，N-末端区域(InaQ-N)单独负责蛋白质的转运过程以及与外膜的结合，而C-末端区域在蛋白质运输中不起作用。低浓度的异丙基-β-D-硫代半乳糖苷(0.1mmoL/L)可诱导细胞膜转运和表面结合能力增强，但培养温度(15℃~37℃)对膜转运和表面结合能力无明显影响。此外，通过构建含有单个InaQ-N的GFP融合蛋白，以及两个和三个成对排列的InaQ-N分子，通过Western印迹分析、免疫荧光显微镜、亚细胞组分的GFP特异性荧光强度分析和流式细胞仪分析，比较了这些蛋白的转运和膜结合活性，结果表明，InaQ-N重复序列的增加导致了表面固定化效率的协同提高。因此，本研究结果可以作为改进基于INP的细胞表面显示系统性能的分子基础。
The ice nucleation protein (INP) of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized. The deduced amino acid sequence of InaQ shares similar protein domains with three P. syringae INPs, namely, InaK, InaZ, and InaV, which were identified as an N-terminal domain, a central repeating domain, and a C-terminal domain. The expression of the full-length InaQ and of various truncated variants was induced in Escherichia coli to analyze their transmembrane transport and surface-binding activities, while using the green fluorescence protein (GFP) as the fusion partner. With two transmembrane segments and a weak secretion signal, the N-terminal domain (InaQ-N) alone was found to be responsible for the transport process as well as for the binding to the outer membrane, whereas the C-terminal region was nonfunctional in protein transport. Increased membrane transport and surface-binding capacities were induced by a low isopropyl-β-D-thiogalactoside concentration (0.1 mmol/l) but not by culture temperatures (15 ºC to 37 ºC). Furthermore, by constructing the GFP-fused proteins with a single InaQ-N, as well as two and three tandemly aligned InaQ-N molecules, the transport and membrane-binding activities of these proteins were compared using Western blot analysis, immmunofluorescence microscopy, and assays of the GFP specific fluorescence intensity of subcellular fractions and flow cytometry, which showed that the increase of InaQ-N repeats resulted in a coordinated increase of the surface-immobilization efficiency. Therefore, the results of this study can serve as a molecular basis for improving the performance of INP-based cell surface-display systems.
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影响因子：
3.5
作者：
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通讯作者：
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影响因子：
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作者：
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