Lactose Carrier Protein of Escherichia coli Structure and Expression of Plasmids Carrying the Y Gene of the lac Operon

Abstract

The previously described hybrid plasmid pC7, which carries lacI+ O+ .DELTA.(Z)Y+ A+ on a 12.3 .times. 106-MW DNA fragment, was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to .dwnarw. d(A-A-T-T) and ligated to the vector pBR322. lacY-carrying inserts of various sizes (MW 1.5-4.7 .times. 106) were obtained. Hybrid plasmid pTE18 (2300-base-pair insert) carries part of the I (repressor) gene, the promotor-operator region, part of the Z (.beta.-galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18-harboring strains, the Y-gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12-16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.