Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of β-galactosidase
Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of β-galactosidase
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DOI：
10.1139/o2012-006
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发表时间：
2012-08-01
期刊：
影响因子：
--
通讯作者：
Sterner, Reinhard
中科院分区：
文献类型：
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作者：
Craig, Douglas B.;Schwab, Thomas;Sterner, Reinhard
对野生型大肠杆菌lacZ进行易错PCR，以产生两个质粒编码的基因文库，其含有约2.6（SD 1.9）个核苷酸交换，导致1.8（SD 1.4）个氨基酸取代。将该文库与含有野生型lacZ的质粒一起沿着用于转化E.大肠杆菌lacZ基因缺失。通过蓝色/白色筛选鉴定表达功能性β-半乳糖苷酶的细胞。使用基于毛细管电泳激光诱导荧光的方案对含有异质诱变的β-半乳糖苷酶群体的细胞裂解物进行单分子测定。随机诱变和野生型酶群体之间的平均催化速率没有显着差异。此外，错误率和群体催化率的方差之间没有明确的模式。这表明，随机序列错误不是这种酶的催化异质性的实质性来源。
Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional beta-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized beta-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.