Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli.
The gene for the soluble cytochrome b562 from Escherichia coli B has been cloned on a SalI fragment. The analysis of the gene reveals the presence of a leader sequence in front of the sequence encoding the mature protein. Expression of cytochrome b562 using the lac-promoter produced the protein to a level of 3-5% of total protein. This over-production enables employment of a simple, high-yield purification protocol to obtain homogeneous cytochrome b562. Spectroscopic and N-terminal sequence analyses of the purified protein demonstrate that it is identical to the chromosomally expressed cytochrome b562 purified and characterized from E. coli B [Itagaki, E. & Hager, L.P. (1966) J. Biol. Chem. 241, 3687-3695]. It is demonstrated that the genomic sequence codes for a classic N-terminal signal sequence and that mature cytochrome b562 is translocated to the periplasmic space.
Nikkila
H
H
Department of Biochemistry, University of Illinois, Urbana 61801.
Gennis
R B
RB
Sligar
S G
SG
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids
D003573:Cytochrome b Group; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C017850:cytochrome b562, E coli