Protein expression and purification
Maintenance of the expression plasmid
Poor plasmid maintenance in the cells can lead to low expression levels. Ampicillin is an unstable antibiotic and is rapidly depleted in growing cultures due in part to the -lactamase secreted by resistant bacterial cells.
It is important to check plasmid levels by plating cells from the expression culture on plates with and without ampicillin. If the stability of the expression construct is a problem, the cultures should be grown in the presence of 200 µg/ml ampicillin, and the level should be maintained by supplementing ampicillin during long growth periods. Alternatively, the cultures may be grown in the presence of carbenicillin, a more stable -lactam, at 50 µg/ml (see Antibiotics in the DNA section of this Protocols and Applications Guide).