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

Mustak A. Kaderbhai, Syed K. Abbas, Naheed N. Kaderbhai, David J. Hopper, Kalim M. Akhtar

Research output: Contribution to journalArticlepeer-review

3 Citations (SciVal)

Abstract

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 (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.
Original languageEnglish
Pages (from-to)4727-4731
Number of pages5
JournalApplied and Environmental Microbiology
Volume69
Issue number8
DOIs
Publication statusPublished - 01 Aug 2003

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