Heterologous Expression in Pseudomonas aeruginosa and Purification of the 9.2-kDa c-Type Cytochrome Subunit of p-Cresol Methylhydroxylase Ciara ´n N. Cronin 1 and William S. McIntire Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121; and Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 Received November 1, 1999, and in revised form January 25, 2000 The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overex- pressed in recombinant form in Pseudomonas aerugi- nosa 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 rel- atively 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 chromatofocus- ing on PolyBuffer Exchanger 94. Purified PchC was ob- tained in a yield of about 50% and was shown to be identical to that resolved from the native flavocyto- chrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes. © 2000 Academic Press p-Cresol methylhydroxylase (EC 1.17.99.1; PCMH) 2 from Pseudomonas putida NCIMB 9869 is a periplas- mic enzyme that catalyzes the transformation of p- cresol to p-hydroxybenzyl alcohol and subsequently to p-hydroxybenzaldehyde. The enzyme is a  2  2 flavocy- tochrome composed of two 9.2-kDa c -type cytochrome subunits (PchC) and two 58.7-kDa flavoprotein sub- units (PchF). Each of the latter subunits has flavin adenine dinucleotide (FAD) covalently attached via a 8-O-tyrosyl-FAD linkage to Tyr384 (1,2). PCMH has been extensively studied in this laboratory, including its isolation (3), determination of the steady-state ki- netic mechanism (4,5), and the resolution of the tet- ramer into its subunits by isoelectric focusing (3,6). The reassembly of the holoenzyme, including the self- catalytic covalent attachment of the FAD, is achieved simply by mixing apoPchF and PchC in the presence of FAD (7). In addition, the complete nucleotide se- quences of both subunits have been determined (2,8) and the high-resolution three-dimensional structures of the holoenzyme with and without bound p-cresol have been solved (9). This structural knowledge has allowed for the development of a proposed catalytic mechanism for the enzyme which involves the partici- pation of selected amino acyl side groups (9). In order to test the accuracy of the proposed mechanism, it is intended in future studies to replace these residues systematically by site-directed mutagenesis and to study the effects of the substitutions on the properties of the enzyme. Toward these goals, the PchF subunit has been over- produced independent of the cytochrome subunit in Escherichia coli, and about 25 mg may be obtained in purified form from 6 liters of cell culture (10). The recombinant flavoprotein is obtained in its dimeric state with FAD noncovalently bound, which may be removed by hydroxyapatite chromatography (10). The production of the PchC cytochrome subunit in recom- binant form has proven, however, to be problematic. 1 To whom correspondence should be addressed at Veterans Af- fairs Medical Center (151-S), 4150 Clement Street, San Francisco, CA 94121. Fax: (415) 750-6959. E-mail: ciaran@itsa.ucsf.edu. 2 Abbreviations used: EDTA, ethylenediaminetetraacetate; FAD, flavin adenine dinucleotide; IPTG, isopropyl-1-thio--D-galactopy- ranoside; LB, Luria–Bertani; ORF, open reading frame; PES, phena- zine ethosulfate; PBE, polybuffer exchanger; PchC, cytochrome sub- unit of PCMH; PchF, flavoprotein subunit of PCMH; PCMH, p-cresol methylhydroxylase. Protein Expression and Purification 19, 74 – 83 (2000) doi:10.1006/prep.2000.1218, available online at http://www.idealibrary.com on 74 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.