IMPROVED BACTERIAL EXPRESSION OF THE HUMAN P-FORM PHENOLSULFOTRANSFERASE - APPLICATIONS TO DRUG-METABOLISM

Bacterial expression of human phenol phenolsulfotransferase (P-PST) ha s provided the opportunity to understand better the catalytic properti es and biological role of this enzyme. However, as the yield of pure p rotein from the currently used expression system was low, we subcloned the P-PST c-DNA into pET-15b, a vector containing an oligohistidine d omain, for improved expression. The fusion protein, His-P-PST, was iso lated from the bacterial cytosol in a single affinity chromatography s tep, using a Ni2+ agarose column. The yield of His-P-PST from the pET- 15b vector was improved 12-fold, compared with P-PST from the original vector. The purity was >99%, as established by sodium dodecyl sulfate -polyacrylamide gel electrophoresis and densitometry scanning. The enz yme was stable for at least 3 weeks when stored in 20% glycerol at -80 degrees C. A very rapid deterioration of the enzyme during 37 degrees C incubations was effectively prevented by the addition of bovine ser um albumin. The sulfonation of several substrates was very similar for His-P-PST and P-PST, with V-max/K-M values (first order rate constant s) for the high-affinity substrate p-nitrophenol of 143+/-27 and 120+/ -25 ml min(-1) mu g(-1) PST [mean+/-SE; not significant (NS)], respect ively, and for the low-affinity substrate acetaminophen of 0.21+/-0.11 and 0.14+/-0.07 ml min(-1) mu g(-1) PST (NS). The V-max/K-M for the s ulfonation of the isoproterenol enantiomers showed a (+)/(-)-enantiome r ratio of 6.2 for His-P-PST and 7.4 for P-PST. Interestingly, 3- to 1 0-fold higher apparent K-M values were obtained for these substrates w ith the crude human liver cytosol, compared with the recombinant P-PST s, suggested to be due to endogenous or dietary P-PST inhibitors in th e liver. In addition, the inhibition of acetaminophen sulfonation by q uercetin was very similar for His-P-PST and P-PST, with IC50 values of 0.10+/-0.03 and 0.05+/-0.01 mu M (NS), respectively. The additional a mino acid residues in the His-P-PST, compared with the recombinant P-P ST, thus did not significantly alter the catalytic properties. This ba cterial expression system should lend itself to routine use in studies of the metabolism of drugs and environmental chemicals.