Jin H J
Department of Genetic Engineering, College of Natural Science, The University of Suwon, Korea.
Mol Cells. 1999 Jun 30;9(3):252-7.
The Erm family of methyltransferases confers the MLS antibiotic resistance to pathogenic microorganism through the mono- or dimethylation of a single adenine residue in 23S rRNA, which is known as the target site for modification. One of the erm genes, ermSF was cloned from Streptomyces fradiae NRRL 2702 by PCR and overexpressed in E. coli BL21(DE3) as both a soluble protein and insoluble aggregate (inclusion body) using the T7 promoter driven expression vector, pET23b. Even though most of the overexpressed protein existed as an inclusion body, E. coli cells showed resistance to erythromycin. The lowering of incubation temperature from 37 degrees C to 22 degrees C facilitated the purification of the protein by increasing the fraction of soluble protein. The soluble protein was purified using immobilized metal ion (Ni2+) affinity chromatography in a one-step manner to the apparent homogeneity. The 23S rRNA of E. coli was found to be a good substrate for the purified ErmSF.
Erm甲基转移酶家族通过对23S rRNA中单个腺嘌呤残基进行单甲基化或二甲基化，赋予致病微生物对大环内酯-林可酰胺-链阳菌素B（MLS）抗生素的抗性，该残基就是已知的修饰靶位点。erm基因之一ermSF通过PCR从弗氏链霉菌NRRL 2702中克隆出来，并使用T7启动子驱动的表达载体pET23b在大肠杆菌BL21(DE3)中作为可溶性蛋白和不溶性聚集体（包涵体）进行过表达。尽管大多数过表达的蛋白以包涵体形式存在，但大肠杆菌细胞仍表现出对红霉素的抗性。将培养温度从37℃降至22℃，通过增加可溶性蛋白的比例促进了蛋白的纯化。可溶性蛋白通过固定化金属离子（Ni2+）亲和色谱一步纯化至表观均一性。发现大肠杆菌的23S rRNA是纯化的ErmSF的良好底物。
