Melibiose permease and alpha-galactosidase of Escherichia coli: Identification by selective labeling using a T7 RNA polymerase/promoter expression system
- Commissariat a l'Energie Atomique, Villefranche-sur-Mer (France)
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. Following generation of a novel 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 melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested.
- OSTI ID:
- 6747175
- Journal Information:
- Biochemistry; (USA), Vol. 29:3; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ESCHERICHIA COLI
GENE REGULATION
GALACTOSIDASE
GENES
PHOSPHOTRANSFERASES
AMINO ACID SEQUENCE
CATTLE
CHEMICAL COMPOSITION
DNA
LABELLING
METHIONINE
RNA POLYMERASES
SULFUR 35
TRACER TECHNIQUES
AMINO ACIDS
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
DRUGS
ENZYMES
EVEN-ODD NUCLEI
GLYCOSYL HYDROLASES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
O-GLYCOSYL HYDROLASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
RUMINANTS
SULFUR ISOTOPES
TRANSFERASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques