Dube D K, Loeb L A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Biochemistry. 1989 Jul 11;28(14):5703-7. doi: 10.1021/bi00440a001.
We have remodeled the gene coding for beta-lactamase by replacing DNA at the active site with random nucleotide sequences. The oligonucleotide replacement (Phe66XXXSer70XXLys73) preserves the codon for the active serine-70 but also contains 15 base pairs of chemically synthesized random sequences that code for 2.5 x 10(6) amino acid substitutions. From a population of Escherichia coli infected with plasmids containing these random inserts, we have selected seven new active-site mutants that render E. coli resistant to carbenicillin and a series of related analogues. Each of the new mutants contains multiple nucleotide substitutions that code for different amino acids surrounding serine-70. Each of the mutants exhibits a temperature-sensitive beta-lactamase activity. This technique offers the possibility of constructing alternative active sites in enzymes on the basis of biological selection for functional variants.
我们通过用随机核苷酸序列替换活性位点的DNA，对β-内酰胺酶的编码基因进行了改造。寡核苷酸替换（Phe66XXXSer70XXLys73）保留了活性丝氨酸-70的密码子，但也包含15个化学合成的随机序列碱基对，这些碱基对编码2.5×10⁶个氨基酸替代。从感染了含有这些随机插入片段质粒的大肠杆菌群体中，我们筛选出了七个新的活性位点突变体，这些突变体使大肠杆菌对羧苄青霉素及一系列相关类似物具有抗性。每个新突变体都包含多个核苷酸替换，这些替换编码丝氨酸-70周围不同的氨基酸。每个突变体都表现出温度敏感的β-内酰胺酶活性。这项技术为基于功能变体的生物学选择在酶中构建替代活性位点提供了可能性。
