Hülsmann K H, Quaas R, Georgalis Y, Saenger W, Hahn U
Institut für Kristallographie, Freie Universität Berlin, F.R.G.
Gene. 1991 Feb 1;98(1):83-8. doi: 10.1016/0378-1119(91)90107-m.
We constructed a semisynthetic gene encoding a DNA-adenine-methyltransferase (Dam) that codes for the same amino acid sequence as the wild type (wt) Escherichia coli dam gene. Since for unknown reasons the entire wt sequence, from the start codon to the end of the gene, could not be cloned, a gene was constructed consisting of a chemically synthesized 5' portion and a 3' portion from the E. coli chromosome. Introduction of this semisynthetic gene into a suitable vector allows overproduction of E. coli Dam in mg amounts per liter E. coli culture, with optimum expression of the gene in the vector pJLA503. This plasmid places the target gene under control of the strong, tandemly arranged pR pL promoters from bacteriophage lambda, regulated by a temperature-sensitive lambda repressor. A rapid, two-column purification protocol is described that allows for very fast purification of the protein. The 32-kDa recombinant protein methylates the sequence GATC.
我们构建了一个编码DNA腺嘌呤甲基转移酶（Dam）的半合成基因，该基因编码的氨基酸序列与野生型（wt）大肠杆菌dam基因相同。由于未知原因，无法克隆从起始密码子到基因末端的整个wt序列，因此构建了一个由化学合成的5'部分和来自大肠杆菌染色体的3'部分组成的基因。将这个半合成基因导入合适的载体中，每升大肠杆菌培养物能够过量产生毫克级的大肠杆菌Dam，并且该基因在载体pJLA503中表达最佳。该质粒将靶基因置于来自噬菌体λ的强串联排列的pR pL启动子的控制之下，由温度敏感的λ阻遏物调节。本文描述了一种快速的双柱纯化方案，可实现蛋白质的极快速纯化。这种32 kDa的重组蛋白可使GATC序列发生甲基化。
