Isolation of extracellular protein from greenhouse soil
Isolation of extracellular protein from greenhouse soil
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DOI：
10.1016/s0038-0717(03)00087-7
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发表时间：
2003-05-01
影响因子：
9.7
通讯作者：
Yonebayashi, K
中科院分区：
文献类型：
--
作者：
Murase, A;Yoneda, M;Yonebayashi, K
虽然细胞外蛋白质在土壤环境中可能发挥重要作用，但这些蛋白质很难分离，因为它们会立即被土壤微生物降解，或与粘土矿物和腐殖物质结合。我们开发了一种从温室土壤中分离胞外蛋白的方法。磷酸盐缓冲液（pH 6.0）用于从土壤中提取蛋白质。不推荐使用pH值较高的磷酸盐缓冲液，因为它提取了大量的非蛋白质有机物以及蛋白质，因此，难以通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离提取的蛋白质。通过过滤除去细胞后，通过用5%三氯乙酸（TCA）沉淀回收溶解在土壤提取物中的蛋白质，并通过SDS-PAGE分离。在32个来自不同温室的土壤样品中检测到10个蛋白质，蛋白质条带的表观分子量范围为35至68 kDa，这表明来自温室培养的一些土壤含有大量可溶于67 mM磷酸盐缓冲液（pH 6.0）的特定蛋白质。其中一个蛋白质的N-末端氨基酸序列被发现是由一种嗜热真菌腐质霉属（Humicola）产生的耐热纤维素酶的同系物。(C)2003爱思唯尔科技有限公司版权所有。
Although extracellular proteins may play an important role in the soil environment, these proteins are difficult to isolate because they are immediately degraded by soil microbes, or become associated with clay mineral and humic substances. We developed a method of isolating extracellular proteins from greenhouse soils. Phosphate buffer (pH 6.0) was used to extract protein from soil. A phosphate buffer with higher pH was not recommended because it extracted a large amount of non-proteinaceous organic matter as well as protein and, as a result, the extracted protein was difficult to separate by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After removing cells by filtration, proteins dissolved in the soil extract were recovered by precipitation with 5% trichloroacetic acid (TCA) and isolated by SDS-PAGE. Proteins were detected in 10 of 32 soil samples derived from different greenhouses and the protein bands ranged in apparent molecular mass from 35 to 68 kDa, suggesting that some of soils derived from greenhouse culture contained significant amounts of a specific protein soluble in 67 mM phosphate buffer (pH 6.0). N-terminal amino acid sequence of one of the isolated proteins was found to be a homologue of thermostable cellulase produced by the genus Humicola, a thermophilic fungus. (C) 2003 Elsevier Science Ltd. All rights reserved.