PRODUCTION AND PURIFICATION OF RECOMBINANT PENTACHLOROPHENOL 4-MONOOXYGENASE AS A GLUTATHIONE S-TRANSFERASE FUSION PROTEIN

Hong Wang, Marja Tiirola, Jaakko Puhakka* and Markku S. Kulomaa, Dept. of Biological and Environmental Science, University of Jyväskylä, PO Box 35, FIN-40351 Jyväskylä, and * Dept. of Environmental Technology, Tampere University of Technology, PO Box 541, FIN-33101 Tampere, Finland.

Pentachlorophenol 4-monooxygenase (PCP-4MO) is the enzyme to initiate degradation of pentachlorophenol (PCP). It catalyzes conversion of PCP to 2,3,5,6-tetrachlorohydroquinone (TeCH) in the presence of NADPH and oxygen. In order to initiate crystallization and studies on structure-function relationship, PCP-4MO was fused with glutathione S-transferase (GST) in an expression vector pGEX-pcpB (deriving from pGEX-3X) and produced in E. coli JM109 cells. The first construct contained a cleavage site for the protease factor Xa. This fusion strategy have allowed one-step purification of the fusion protein (GST-PCP-4MO) from crude bacterial extracts under nondenaturing conditions by affinity chromatography on a glutathione Sepharose 4B column and also facilitated simple proteolytic cleavage of the fusion tag. Cleavage of the GST-PCP-4MO by factor Xa worked, but the recombinant PCP-4MO (rePCP-4MO) had a double band on SDS-PAGE suggesting a non-specific digestion within rePCP-4MO. In addition, factor Xa remained with the released rePCP-4MO and should have been removed one way or the other. Crystallization requires however both pure and homogenious protein preparation. To facilitate this, another cleavage site was introduced between the GST and rePCP-4MO. A seven-amino-acid (Glu-Lys-Leu-Tyr-Phe-Gln-Gly) cleavage site is specific for tobacco etch virus (TEV) protease and it was located immediately after the factor Xa cleavage site in the pGEX-pcpB plasmid. This construct encoded an open reading frame for a GST/factor Xa cleavage site/TEV cleavage site/PCP-4MO fusion protein. In addition, a recombinant TEV protease containing a polyhistidine tag at the N-terminus (reTEV-HIS) was used for PCP-4MO cleavage, which allows efficient removal of the His-tagged protease from rePCP-4MO on a Ni2+-column.

The GST-PCP-4MO fusion protein was produced by an overnight incubation at room temperature. It could efficiently be purified to homogeneity from the crude lysate in one-step on a glutathione Sepharose 4B column in the presence of Sarkosyl and Triton X-100. The yield of the fusion protein was 10-15mg/l. Incubation of the fusion protein with reTEV-HIS protease caused highly specific and efficient release of the rePCP-4MO. The cleavage mixture was passed over a glutathione Sepharose 4B column to remove GST and then over a Ni2-column to remove TEV protease. SDS-PAGE electrophoresis showed a single 63KDa band identical to the PCP-4MO. In immunoblot analysis, the band was detected with a specific antibody raised against the rePCP-4MO. Identity of the rePCP-4MO was further confirmed by N-terminal amino acid sequencing. In order to analyze activity of the rePCP-4MO, PCP was used as substrate and the enzymatic activity was assayed by monitoring concentration of PCP and TeCH in the reaction mixture. A 20Ál aliquot was analyzed by an HPLC procedure. The HPLC chromatograms showed significant increase in concentration of TeCH and decrease in that of PCP after adding rePCP-4MO to the reaction mixture. The results indicated that the rePCP-4MO produced as a GST-fusion protein by a bacterial expression system was structurally very similar or identical to the native PCP-4MO. After cleavage by a TEV protease, significant amount of enzymatic activity could be detected indicating proper folding of the rePCP-4MO.

In conclusion, the results indicate that we have an efficient expression system for production of the rePCP-4MO to be used in studies of 3D-structure by x-ray crystallography and of structure-function relationship by site-directed mutagenesis.