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 (EIImtl) and its upstream promoter region (Pmtl) were subcloned approximately 150 base pairs downstream of a lambda PR promoter on a multicopy mutagenesis/expression vector and used to transform a mutant (MtlA-) E. coli strain. Induction at 42 degrees C led to 50 to 100-fold overproduction of EIImtl (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 EIImtl compared with mannitol-induced wild-type levels.
van Weeghel
R P
RP
Department of Physical Chemistry, University of Groningen, The Netherlands.
Keck
W
W
Robillard
G T
GT
D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005838:Genotype; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins; D010641:Phenotype; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli