Roberts J D, McMacken R
Nucleic Acids Res. 1983 Nov 11;11(21):7435-52.
The bacteriophage lambda O protein participates in the initiation of lambda DNA replication. The lambda O gene was cloned into plasmid pKC30 such that its expression was controlled by the lambda PL promoter. A lambda prophage-coded thermosensitive cI repressor was used to regulate transcription of the cloned O gene. Thermal inactivation of the lambda cI repressor resulted in overproduction of the O protein until it constituted approximately 20% of the total cellular protein of Escherichia coli. A simple three-step purification protocol was developed that yields several milligrams of homogeneous O protein per gram of cell paste. The precise position of the O gene in the known lambda DNA sequence was identified from the amino-terminal sequence of the isolated O protein. Purified O protein stimulated the replication of plasmid lambda dv DNA in vitro and specifically bound to duplex DNA fragments carrying the lambda replication origin.
噬菌体λO蛋白参与λDNA复制的起始过程。将λO基因克隆到质粒pKC30中，使其表达受λPL启动子控制。利用λ原噬菌体编码的温度敏感型cI阻遏物来调节克隆的O基因的转录。λcI阻遏物的热失活导致O蛋白过量产生，直至其占大肠杆菌总细胞蛋白的约20%。开发了一种简单的三步纯化方案，每克细胞糊可产生数毫克的纯O蛋白。从分离的O蛋白的氨基末端序列确定了O基因在已知λDNA序列中的精确位置。纯化的O蛋白在体外刺激质粒λdv DNA的复制，并特异性结合携带λ复制起点的双链DNA片段。
