REGULATED HIGH-LEVEL EXPRESSION OF THE MANNITOL PERMEASE OF THE PHOSPHOENOLPYRUVATE-DEPENDENT SUGAR PHOSPHOTRANSFERASE SYSTEM IN ESCHERICHIA-COLI

The structural gene (mtlA) of the Escherichia coli phosphoenolpyruvate-dependent mannitol-transport protein (EII(mtl)) and its upstream promoter region (P(mtl)) were subcloned ~ 150 base pairs downstream of a λ P(R) promoter on a multicopy mutagenesis/expression vector and used to transform a mutant (MtlA-) E. coli strain. Induction at 42°C led to 50- to 100-fold overproduction of EII(mtl) (5-10 mg/g of cell wet weight) relative to mannitol-induced levels in a wild-type (Mtl+) strain. Most of the overproduced protein was sequestered as an inactive form in inclusion bodies and cytoplasmic membranous structures. The protein could be extracted in an active form by rupturing the cells with lysozyme and sonication or with a passage through a French pressure cell and incubating the inclusion bodies and membranous structures with detergent (Lubrol PX or deoxycholate) in the presence of Q or S Sepharose ion-exchange resin for several hours. This procedure resulted in a 20- to 25-fold overproduction of active EII(mtl) compared with mannitol-induced wild-type levels.