FORMAMIDOPYRIMIDINE-DNA GLYCOSYLASE OF ESCHERICHIA-COLI - CLONING AND SEQUENCING OF THE FPG STRUCTURAL GENE AND OVERPRODUCTION OF THE PROTEIN
FORMAMIDOPYRIMIDINE-DNA GLYCOSYLASE OF ESCHERICHIA-COLI - CLONING AND SEQUENCING OF THE FPG STRUCTURAL GENE AND OVERPRODUCTION OF THE PROTEIN
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DOI：
10.1002/j.1460-2075.1987.tb02629.x
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发表时间：
1987-10-01
期刊：
影响因子：
11.4
通讯作者：
LAVAL, J
中科院分区：
文献类型：
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作者：
BOITEUX, S;OCONNOR, TR;LAVAL, J
使用质粒pBR322作为载体构建了由大DNA片段(10-15kb)组成的大肠杆菌基因组文库。从其中单独筛选了 700 个克隆，以增加 N7-甲基鸟嘌呤（2-6-二氨基-4-羟基-5N-甲基-甲酰胺嘧啶）或 Fapy 开环形式的切除。与野生型菌株相比，一个克隆过量产生了 10 倍的 Fapy-DNA 糖基化酶活性。 Fapy-DNA 糖基化酶过量生产者特征与 15 kb 重组质粒 (pFPG10) 相关。亚克隆后，将含有 Fapy-DNA 糖基化酶基因 (fpg+) 的 1.4-kb 片段插入质粒 pUC18 和 pUC19 中，分别产生 pFPG50 和 pFPG60。携带 pFPG60 的细胞的糖基酶活性增加了 50 至 100 倍，并过表达 31 kd 的蛋白质。从这些细胞中，Fapy-DNA 糖基化酶被纯化至明显的物理同质性，如 SDS 聚丙烯酰胺凝胶上 32 kd 处的单个蛋白质条带所证明的那样。蛋白质的氨基酸组成和从核苷酸序列推导的氨基酸序列证明克隆片段含有编码Fapy-DNA糖基化酶的结构基因。 fpg基因的核苷酸序列由809个碱基对组成，编码269个氨基酸的蛋白质，计算摩尔数为1。重量 30.2 kd。
An Escherichia coli genomic library composed of large DNA fragments (10-15 kb) was constructed using the plasmid pBR322 as vector. From it 700 clones were individually screened for increased excision of the ring-opened form of N7-methylguanine (2-6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine) or Fapy. One clone overproduced the Fapy-DNA glycosylase activity by a factor of 10-fold as compared with the wild-type strain. The Fapy-DNA glycosylase overproducer character was associated with a 15-kb recombinant plasmid (pFPG10). After subcloning a 1.4-kb fragment which contained the Fapy-DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively. The cells harbouring pFPG60 displayed a 50- to 100-fold increase in glycosylase activity and overexpressed a 31-kd protein. From these cells the Fapy-DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 32 kd on SDS-polyacrylamide gels. The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy-DNA glycosylase. The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol. wt of 30.2 kd.