用于分离膜脂合成酶的基因克隆：大肠杆菌中磷脂酰丝氨酸合酶的过量表达
Gene cloning for the isolation of enzymes of membrane lipid synthesis: phosphatidylserine synthase overproduction in Escherichia coli.
作者信息
Raetz C R, Larson T J, Dowhan W
出版信息
Proc Natl Acad Sci U S A. 1977 Apr;74(4):1412-6. doi: 10.1073/pnas.74.4.1412.
Abstract
We have screened a bank of 2000 E. coli strains carrying hybrid ColE1 plasmids [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] for those that correct the temperature sensitivity of a mutant in CDP-1,2-diacyl sn-glycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase). Two hybrid plasmids of this kind (pLC34-44 and pLC34-46) were identified and characterized. Strains carrying these plasmids overproduce the synthase by 6- to 15-fold, as demonstrated by assays of extracts and purification to homogeneity of the overproduced enzyme. The overproduced synthase, like the wild-type enzyme, is found associated predominately with the ribosomal fraction of crude cell extracts. Because the membrane phospholipid composition of these overproducers is not greatly altered, we suggest that the synthase is normally present in excess.
摘要
我们从一组含有杂交ColE1质粒的2000株大肠杆菌菌株库中进行筛选[克拉克，L.和卡尔本，J.（1976年）《细胞》9卷，91 - 99页]，寻找能够校正CDP - 1,2 - 二酰基sn - 甘油：L - 丝氨酸O - 磷脂酰转移酶（EC 2.7.8.8，磷脂酰丝氨酸合酶）突变体温度敏感性的菌株。鉴定并表征了两种此类杂交质粒（pLC34 - 44和pLC34 - 46）。通过对提取物的测定以及对过量表达的酶进行纯化直至均一性，结果表明携带这些质粒的菌株使合酶的产量过量6至15倍。与野生型酶一样，过量表达的合酶主要存在于粗细胞提取物的核糖体部分。由于这些过量表达菌株的膜磷脂组成没有太大改变，我们认为合酶通常是过量存在的。
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