Sugiyama A, Kato H, Nishioka T, Oda J
Institute for Chemical Research, Kyoto University.
Biosci Biotechnol Biochem. 1992 Mar;56(3):376-9. doi: 10.1271/bbb.56.376.
Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.
利用质粒pUNAd37（pUC18的衍生物）在大肠杆菌中实现了编码天冬酰胺合成酶（EC 6.3.1.1）的大肠杆菌K-12 asnA基因的过表达。该质粒是通过优化启动子和核糖体结合区域之间的DNA序列构建而成的。该酶约占大肠杆菌细胞中总可溶性蛋白的15%，通过DEAE-纤维素亲和柱色谱和蓝色-纤维素亲和柱色谱很容易纯化至表观均一。纯化蛋白的氨基末端序列、氨基酸组成和分子量与基于该基因DNA序列预测的值一致。此外，通过凝胶过滤测得的天然分子量证实天冬酰胺合成酶以相同亚基的二聚体形式存在。
