Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase.
The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recently developed pUCP-Nde vector. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, were generally unsuccessful, yielding relatively low levels of the protein. In contrast, the mature form of PchC accumulated in the periplasmic space of P. aeruginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enriched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediaminetetraacetate in the presence of sucrose. The cytochrome was purified to homogeneity from the periplasmic extract by anion-exchange chromatography on DEAE-Sepharose CL-6B followed by chromatofocusing on PolyBuffer Exchanger 94. Purified PchC was obtained in a yield of about 50% and was shown to be identical to that resolved from the native flavocytochrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes.
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Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA. ciaran@itsa.ucsf.edu
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D020134:Catalytic Domain; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C049502:4-cresol dehydrogenase (hydroxylating)