Melibiose permease and .alpha.-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system
Identification and selective labeling of the melibiose permease and .alpha.-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel for EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that .alpha.-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound milibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al, (1984) J. Biol. Chem. 259, 1807].