A cytochrome c from a lupanine-transforming Pseudomonas putida strain is expressed in Escherichia coli during aerobic cultivation and efficiently exported and assembled in the periplasm.
We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain. Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm ( approximately 4 mg of hemoprotein/liter of culture). The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine. Comparison of the N-terminal amino acid sequence of the isolated cytochrome c with that deduced from the DNA sequence indicated that the signal sequence was processed at the bond position predicted by the SigPep program. The molecular size of the cytochrome c determined by electrospray mass spectrometry (9,595) was in precise agreement with that predicted from the nucleotide sequence.
Kaderbhai
Mustak A
MA
Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DD, Wales, United Kingdom. mak@aber.ac.uk
Hopper
David J
DJ
Akhtar
Kalim M
KM
Abbas
Syed K
SK
Kaderbhai
Naheed N
NN
D000332:Aerobiosis; D000470:Alkaloids / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D019897:Periplasm / Q000201:enzymology*; D021381:Protein Transport; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013034:Sparteine / Q000031:analogs & derivatives
D000470:Alkaloids; D001426:Bacterial Proteins; D003574:Cytochrome c Group; D011994:Recombinant Proteins; C083951:cytochrome c553; C008298:lupanine; D013034:Sparteine; C022302:cytochrome C-552