Purification and Properties of Phospholipase A from the Outer Membrane of Overproducing Escherichia Coli K-12
Phospholipase A (PLA) from Escherichia coli K-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 vivo this “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 Geus et al., 1983) for the E. coli K-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.
KeywordsCell Envelope Heat Shock Treatment Phospholipid Turnover Bacterial Cell Envelope Phospholipid Breakdown
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