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-creso l methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpress ed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recent ly developed pUCP-Nde vector, Efforts to produce the cytochrome in Escheric hia coli using a pET vector, with or without its signal peptide, were gener ally unsuccessful, yielding relatively low levels of the protein. In contra st, the mature form of PchC accumulated in the periplasmic space of P. aeru ginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enri ched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediami netetraacetate in the presence of sucrose. The cytochrome was purified to h omogeneity 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 r ecombinant c-type cytochromes. (C) 2000 Academic Press.