Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida

Marianne Hallberg Thuesen, Allan Nørgaard, Anne Merete Hansen, Michael Bjerg Caspersen, Hans Erik Mølager Christensen

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Abstract

The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow clectrospray ionization time-of-flight mass spectrometry, and electrochemistry. Cyclic voltammograms and UV-vis electronic absorption spectra were indistinguishable from the equivalent data of native P. stutzeri cytochrome c(4). Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c(4) were identical. Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure.
Original languageEnglish
JournalProtein Expression and Purification
Volume27
Issue number1
Pages (from-to)175-181
ISSN1046-5928
Publication statusPublished - 2003

Cite this

Thuesen, M. H., Nørgaard, A., Hansen, A. M., Caspersen, M. B., & Christensen, H. E. M. (2003). Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida. Protein Expression and Purification, 27(1), 175-181.