Expression of human dopamine beta-hydroxylase in Drosophila Schneider 2 cells.
Human dopamine beta-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are identical in size (61 kDa). The Km of tyramine for native and recombinant human enzymes are virtually the same but higher than bovine DBH by about 3-fold. Likewise, the inhibition of native and recombinant human DBH by fusaric acid and SKF102698 is not significantly different but IC50 values are 2-3-fold higher than that for the bovine enzyme. These results strongly support the conclusion that recombinant human DBH from Drosophila S2 cells can be used in place of human neuroblastoma-derived DBH for drug screening, characterization of the enzyme's physicochemical properties, and determination of structure-function relationships. The Drosophila expression system has thus provided a convenient source for large quantities of human DBH enzyme.
Biotechnology, Syntex Discovery Research, Palo Alto, CA 94304, USA.
B
B
Li
S
S
Tsing
A H
AH
Kosaka
B
B
Nguyen
E G
EG
Osen
C
C
Bach
H
H
Chan
J
J
Barnett
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002461:Cell Line, Transformed; D004299:Dopamine beta-Hydroxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila / Q000201:enzymology / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism / Q000821:virology; D014162:Transfection; D014407:Tumor Cells, Cultured
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004299:Dopamine beta-Hydroxylase