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Applied and Environmental Microbiology, August 2003, p. 4727-4731, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4727-4731.2003

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,* David J. Hopper, Kalim M. Akhtar, Syed K. Abbas,{dagger} and Naheed N. Kaderbhai

Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DD, Wales, United Kingdom

Received 3 March 2003/ Accepted 13 May 2003

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.

* Corresponding author. Mailing address: Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth SY23 3DD, Wales, United Kingdom. Phone: 44 1970 622294. Fax: 44 1970 622294. E-mail: mak{at}aber.ac.uk.

{dagger} Present address: The School of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

Applied and Environmental Microbiology, August 2003, p. 4727-4731, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4727-4731.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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