Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus
Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus
复制标题
DOI：
10.1128/jb.178.19.5586-5591.1996
复制
发表时间：
1996-10-01
影响因子：
3.2
通讯作者：
Robillard, GT
中科院分区：
文献类型：
--
作者：
Henstra, SA;Tolner, B;Robillard, GT
通过与大肠杆菌EI、HPr和IIA（Mtl）的体外互补，在嗜热脂肪芽孢杆菌中鉴定了甘露醇磷酸转移酶系统（PTS）。根据E.以大肠杆菌和肉葡萄球菌（StaphylococcuscarnosusEII，Mt 1）为材料，通过PCR方法，制备了地高辛标记的探针，用此探针分离出三个7.2kb的重叠DNA片段，分别含有编码甘露醇IICB、调节子IIA和甘露醇-1-磷酸脱氢酶的基因mtlA、mtlR、mtlF和mtlD。该基因全长413 bp，编码471个氨基酸，分子量为50.1 kDa，与S.肉色E. carnosus和E. coli和B.枯草杆菌。该酶在E.将其置于强tac启动子之后，在60 ℃下热灭活B的速率。嗜热脂肪菌IICBMt 1在E. coli比E. coli IICBMt 1，B中的IICBMt 1。嗜热脂肪菌在85 ℃时活性最大，因此非常热稳定。在六个组氨酸融合到转运蛋白的C-末端部分后，在Ni-次氮基三乙酸树脂上将酶纯化至大于95%的纯度。
A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mtl) were used to develop a digoxigenin-labeled probe by PCR, Using this probe, ale isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively, The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa, The amino acid sequence shows high similarity with the sequence of IICBMtl of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter, The rate of thermal inactivation at 60 degrees C of B. stearothermophilus IICBMtl expressed in E. coli was two times lower than that of E. coli IICBMtl, IICBMtl in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.