Cloning, expression, and purification of the general stress protein YhbO from Escherichia coli
Cloning, expression, and purification of the general stress protein YhbO from Escherichia coli
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DOI：
10.1016/j.pep.2005.11.011
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发表时间：
2006-06-01
影响因子：
1.6
通讯作者：
Richarme, Gilbert
中科院分区：
文献类型：
--
作者：
Abdallah, Jad;Kern, Renee;Richarme, Gilbert
我们克隆、表达并纯化了大肠杆菌yfk基因产物，其氨基酸序列与枯草芽孢杆菌总应激蛋白18 （yfk M基因产物）、荧光焦球菌胞内蛋白酶PfpI和人类帕金森病蛋白DJ-1同源。通过聚合酶链反应从大肠杆菌中扩增YhbO基因，得到YhbO编码基因。将其插入表达质粒pET-21a中，在噬菌体T7启动子和lac操作子的转录控制下。用YhbO表达载体pET-21a-yhbO转化的BL21 （DE3）大肠杆菌菌株在SDS-PAGE上积累了大量的可溶性蛋白，其分子量为20 kDa，与预期的YhbO分子量相匹配。通过离子交换层析和羟基磷灰石层析纯化YhbO，并通过n端测序和质谱分析证实其同源性。天然蛋白以单体、三聚体和六聚体形式存在。我们还报道了YhbO与一般应激蛋白YfkM之间有很强的序列同源性（64%的同源性），这表明YhbO是一种应激蛋白，并且YhbO与博氏焦球菌胞内蛋白酶PhpI之间有很强的结构同源性。然而，使用传统的生化底物，我们无法检测YhbO的任何蛋白水解或肽水解活性。(c) 2005爱思唯尔公司版权所有。
We cloned, expressed, and purified the Escherichia coli yhbO gene product, which is an aminoacid sequence homolog to the Bacillus subtilis general stress protein 18 (the yfk M gene product), the Pyrococcus furiosus intracellular protease PfpI, and the human Parkinson disease protein DJ-1. The gene coding for YhbO was generated by amplifying the yhbO gene from E coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21 (DE3) E. coli strain transformed with the YhbO-expression vector, pET-21a-yhbO, accumulates large amounts of a soluble protein with a molecular mass of 20 kDa in SDS-PAGE that matches the expected YhbO molecular weight. YhbO was purified to homogeneity by ion exchange chromatography and hydroxyapatite chromatography, and its identity was confirmed by N-terminal sequencing and mass spectrometry analysis. The native protein exists in monomeric, trimeric, and hexameric forms. We also report a strong sequence homology between YhbO and the general stress protein YfkM (64% identities), which suggests that YhbO is a stress protein, and a strong structural homology between YhbO and the Pyrococcus horikoshii intracellular protease PhpI. We could not, however, detect any proteolytic or peptidolytic activity of YhbO, using classical biochemical substrates. (c) 2005 Elsevier Inc. All rights reserved.