Purification and Properties of Phospholipase A from the Outer Membrane of Overproducing Escherichia Coli K-12
Purification and Properties of Phospholipase A from the Outer Membrane of Overproducing Escherichia Coli K-12
复制标题
过量生产大肠杆菌K-12外膜磷脂酶A的纯化及性质
DOI：
10.1007/978-1-4684-5212-9_23
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
G. Haas
中科院分区：
文献类型：
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作者：
P. D. Geus;N. Riegman;B. Verheij;G. Haas
来自大肠杆菌K-12的磷脂酶A是一种分子量为30kD的蛋白质，位于细菌细胞膜的外膜上。与大多数其他膜结合的磷脂酶一样，它的功能仍不清楚。据推测，这种酶在正常生长的细胞中是不活跃的，因为无法检测到磷脂的周转。然而，磷脂的破裂可以由许多处理方法引起，所有这些处理方法都会扰乱膜的完整性，如洗涤剂、EDTA、酒精和热休克处理。显然，在活体中，这种“激活”现象需要一个非常严格的调节机制，以防止有机体的自我破坏。此外，在自然界中大多数已知的膜结构中几乎矛盾地存在PLAS，这使这成为一个普遍而有趣的问题(见van den Bosch，1980)。为了研究膜结合聚乳酸的这一性质和其他性质，需要相对大量的纯酶。由于其丰度很低(200-500拷贝/细胞；Sandella&Kornberg，1971)，到目前为止，细菌酶的研究受到了严重阻碍。为了绕过这个问题，我们为你克隆了结构基因(de Geuset al.，1983)。ColiK-12PLA，通过操纵之前的转录信号，我们获得了该酶的过量生产200倍。现在，提纯大量的聚乳酸成为可能。在本文中，我们报道了该酶的克隆、过量生产、纯化和性质的一些细节。
Phospholipase A (PLA) fromEscherichia coliK-12 is a protein of 30 Kilodalton (Kd) molecular weight, located in the outer membrane (OM) of the bacterial cell envelope. Its function, as for most other membrane bound phospholipases, is still unknown. Presumably, the enzyme is inactive in normally growing cells, since no phospholipid turnover can be detected. However, phospholipid breakdown can be induced by a number of treatments, all of which perturb membrane integrity,vizdetergent-, EDTA-, alcohol- and heat shock treatment. Obviously,in vivothis “activation”phenomenon requires a very strict regulatory mechanism to prevent self-destruction of the organism. Moreover, the almost paradoxical presence of PLAses in, or at, most of the known membrane structures in nature make this a general and intriguing problem (see for a review, van den Bosch, 1980). In order to study this and other properties of membrane bound PLA, relatively large amounts of pure enzyme are needed. Because of its very low abundance (200–500 copies/cell; Scandella & Kornberg, 1971), work on the bacterial enzyme has, so far, been severely hampered. To circumvent this problem, we have cloned the structural gene (de Geuset al., 1983) for theE. coliK-12 PLA and by manipulating the transcriptional signals which precede it, we have obtained a 200-fold overproduction of the enzyme. Purification of ample amounts of PLA now became possible. In this paper, we report some of the details of the cloning, overproduction, purification, and properties of the enzyme.