Nwankwo D O, Maunus R E, Xu S
New England Biolabs, Inc., Beverly, MA 01915, USA.
Gene. 1997 Jan 31;185(1):105-9. doi: 10.1016/s0378-1119(96)00641-5.
The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.
编码醋酸醋杆菌AatII限制性内切酶和甲基化酶的基因已在大肠杆菌中克隆并表达。测定了aatIIM和aatIIR基因的核苷酸序列。aatIIM和aatIIR基因分别为996 bp和1038 bp，编码预测分子量为36.9 kDa的331个氨基酸的甲基化酶，以及预测分子量为38.9 kDa的345个氨基酸的AatII限制性内切酶。这两个基因重叠4个碱基对，并以相同方向转录。aatIIRM基因位于一个假定的质粒转移基因旁边。构建了一个稳定的高产菌株，其中aatIIM基因由pSC101衍生质粒表达。将aatIIR基因插入到一个经过修饰的T7表达载体中，该载体在T7启动子上游带有转录终止子。通过DEAE琼脂糖、肝素琼脂糖和磷酸纤维素柱色谱法，将重组AatII限制性内切酶纯化至接近均一。
