Wolfes H, Alves J, Fliess A, Geiger R, Pingoud A
Nucleic Acids Res. 1986 Nov 25;14(22):9063-80. doi: 10.1093/nar/14.22.9063.
We have constructed a plasmid (pRIF 309+) carrying the EcoRI restriction endonuclease gene and the f1 origin of replication. Upon transformation of this plasmid into E. coli and infection with bacteriophage f1 single stranded plasmids are produced which can be used for sequencing and site directed mutagenesis. Using this single stranded DNA and synthetic oligodeoxynucleotides we have introduced point mutations at defined positions of the EcoRI gene. Since in pRIF309+ the EcoRI gene is under the control of the pL-promoter, high level expression of the mutated EcoRI gene could be obtained upon induction. Mutant EcoRI enzymes were purified to homogeneity and characterized in structural and functional terms. Our results demonstrate that the Glu 111----Gln, Glu 144----Gln and Arg 145----Lys -mutants adopt a very similar conformation as the wild type enzyme, but have by two orders of magnitude smaller specific activities than the wild type enzyme, mainly due to a reduction of the Vmax-value.
我们构建了一个携带EcoRI限制性内切酶基因和f1复制起点的质粒（pRIF 309+）。将该质粒转化到大肠杆菌中并感染噬菌体f1后，会产生单链质粒，可用于测序和定点诱变。利用这种单链DNA和合成的寡脱氧核苷酸，我们在EcoRI基因的特定位置引入了点突变。由于在pRIF309+中EcoRI基因受pL启动子的控制，诱导后可获得突变EcoRI基因的高水平表达。突变的EcoRI酶被纯化至同质，并从结构和功能方面进行了表征。我们的结果表明，Glu 111→Gln、Glu 144→Gln和Arg 145→Lys突变体与野生型酶具有非常相似的构象，但比野生型酶的比活性小两个数量级，主要是由于Vmax值的降低。
