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High level expression and characterization of recombinant human hippocampus phenol sulfotransferase: a novel phenol-sulfating form of phenol sulfotransferase


High level expression and characterization of recombinant human hippocampus phenol sulfotransferase: a novel phenol-sulfating form of phenol sulfotransferase



Protein Expression and Purification 11(1): 125-134



ISSN/ISBN: 1046-5928

PMID: 9325148

DOI: 10.1006/prep.1997.0782

Phenol sulfotransferases (PSTs) represent a family of sulfotransferase enzymes that modify the biologic activities and excretion of phenolic compounds and monoamines. A novel human hippocampal PST (H-PST) cDNA with homology to phenol (P) and monoamine (M) forms of PST was previously isolated from brain. To compare the biochemical properties of H-PST with that of phenol (P-PST) and monoamine (M-PST) sulfotransferases, high level expression of recombinant H-PST was achieved in this study with the pET3c vector in BL21(DE3) Escherichia coli cells. Expression was demonstrated by isopropyl beta-D-thiogalactopyranoside induction of 34-kDa H-PST that represented 5-10% of total E. coli proteins. Purification by ion-exchange chromatography on DEAE-Sepharose yielded more than 2 mg of H-PST. Characterization showed that H-PST exists as a homodimer of 60-65 kDa by gel filtration chromatography. H-PST prefers p-nitrophenol as substrate and does not sulfate dopamine or neuropeptide substrates. Kinetic studies showed that H-PST possessed K(m(app)) and Vmax(app) values of 3 microM p-nitrophenol and 160 nmol/min/mg, respectively. H-PST was sensitive to inhibition by DCNP (2,6-dichloro-4-nitrophenol). H-PST is thermolabile since its activity was reduced upon preincubation at 37 degrees C. These results indicate that H-PST shows similarities and differences compared to P-PST and M-PST sulfotransferases. P-PST prefers p-nitrophenol as substrate, is sensitive to inhibition by DCNP, and is thermostable; in contrast, M-PST prefers monoamines as substrate, is not sensitive to DCNP, and is thermolabile. The distinct profile of biochemical properties of H-PST, and its primary sequence homology to P-PST and M-PST, suggests that H-PST represents a novel allelic variant of human phenol sulfotransferases. Importantly, this study demonstrates that high level expression of H-PST allows determination of distinguishing characteristics of variant forms of PSTs.

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Accession: 008771689

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