Overproduction and high-yield purification of phospholipase A from the outer membrane of Escherichia coli K-12.
The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda. Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild-type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005815:Genetic Code; D006868:Hydrolysis; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification*; D010957:Plasmids; D013194:Staining and Labeling; D013329:Structure-Activity Relationship; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D010740:Phospholipases; D010741:Phospholipases A