[5] Glycerophosphate acyltransferase from Escherichia coli

Add time:08/23/2019    Source:sciencedirect.com

Publisher SummaryThis chapter discusses strategies of purifying and characterizing the glycerophosphate acyltransferase. These are (1) cloning of the plsB structural gene encoding the glycerophosphate acyltransferase and construction of strains, which overproduce the enzyme; (2) effective use of nonionic detergents to solubilize the glycerophosphate acyltransferase from membranes and the development of chromatographic steps, which resolve the solubilized, hydrophobic proteins; and (3) design of a quantitatively efficient reconstitution methodology to restore activity to enzyme preparations, which are stable, but inactive, in the presence of detergent. The plsB structural gene encoding the 83,000 Mr glycerophosphate acyltransferase polypeptide has been cloned by monitoring the ability of DNA from a total E. coli library to overcome the glycerol 3-phosphate growth requirement of a plsB- mutant strain. Purification is divided into three steps of fractionation based on hydrophobic dye interaction, combined sizing and hydrophobic interaction, and ion exchange. The efficiency of 83,000 Mr polypeptide recovery and reconstitution of activity under conditions is tabulated in the chapter.

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