Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida.
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 electrospray 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.
Thuesen
Marianne H
MH
Department of Chemistry, Building 207, Technical University of Denmark, 2800 Lyngby, Denmark.
Nørgaard
Allan
A
Hansen
Anne M
AM
Caspersen
Mikael B
MB
Christensen
Hans E M
HE
D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006418:Heme; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D013057:Spectrum Analysis
D003574:Cytochrome c Group; D011994:Recombinant Proteins; C034186:cytochrome C4; D006418:Heme; D005947:Glucose