Improved bacterial expression of the human P form phenolsulfotransferase. Applications to drug metabolism.
Bacterial expression of human phenol phenolsulfotransferase (P-PST) has provided the opportunity to understand better the catalytic properties and biological role of this enzyme. However, as the yield of pure protein from the currently used expression system was low, we subcloned the P-PST c-DNA into pET-15b, a vector containing an oligohistidine domain, for improved expression. The fusion protein, His-P-PST, was isolated from the bacterial cytosol in a single affinity chromatography step, 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 enzyme 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 serum albumin. The sulfonation of several substrates was very similar for His-P-PST and P-PST, with Vmax/KM values (first order rate constants) for the high-affinity substrate p-nitrophenol of 143 +/- 27 and 120 +/- 25 ml min-1 microgram-1 PST [mean +/- SE; not significant (NS)], respectively, and for the low-affinity substrate acetaminophen of 0.21 +/- 0.11 and 0.14 +/- 0.07 ml min-1 microgram-1 PST (NS). The Vmax/KM for the sulfonation of the isoproterenol enantiomers showed a (+)/(-)-enantiomer ratio of 6.2 for His-P-PST and 7.4 for P-PST. Interestingly, 3- to 10-fold higher apparent KM values were obtained for these substrates with the crude human liver cytosol, compared with the recombinant P-PSTs, suggested to be due to endogenous or dietary P-PST inhibitors in the liver. In addition, the inhibition of acetaminophen sulfonation by quercetin was very similar for His-P-PST and P-PST, with IC50 values of 0.10 +/- 0.03 and 0.05 +/- 0.01 microM (NS), respectively. The additional amino acid residues in the His-P-PST, compared with the recombinant P-PST, thus did not significantly alter the catalytic properties. This bacterial expression system should lend itself to routine use in studies of the metabolism of drugs and environmental chemicals.
Lewis
A J
AJ
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, USA.
Kelly
M M
MM
Walle
U K
UK
Eaton
E A
EA
Falany
C N
CN
Walle
T
T
D015239:Arylsulfotransferase / Q000235:genetics* / Q000378:metabolism; D001419:Bacteria / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004795:Enzyme Stability; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015239:Arylsulfotransferase