Characterization and partial purification of human monoamine oxidase-B expressed in Escherichia coli.
Monoamine oxidases (MAO-A and MAO-B) are enzymes that play a key role in the degradation of endogenous and dietary monoamines. A full-length cDNA of the B-type of MAO, isolated from a human liver cDNA library, was cloned into a prokaryotic expression vector (pET11c). Escherichia coli which was transfected with the recombinant plasmid expressed an insoluble protein product with the expected molecular weight (65 kDa). However, in the inclusion body fraction, where most of the recombinant protein was present, no MAO activity was observed. In contrast, the membrane fraction of the bacterial lysates expressed catalytic activity as estimated by oxidative deamination of beta-phenylethylamine and tyramine. The active enzyme protein was solubilized with Triton X-100 and partly purified (80-fold) on a DEAE-Sepharose column. This enzyme activity showed properties very similar to those of human brain and platelet MAO-B. Moreover, a single band of the expected molecular size was observed on an immunoblot. The peak fraction from the DEAE-Sepharose separation was further purified on a tyramine-Sepharose column, yielding a highly purified enzyme (190-fold), visible as a band on a sodium dodecyl sulfate-containing polyacrylamide gel.
Lu
G
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Department of Medical Pharmacology, Uppsala University, Sweden.
Unge
T
T
Owera-Atepo
J B
JB
Shih
J C
JC
Ekblom
J
J
Oreland
L
L
D001792:Blood Platelets / Q000201:enzymology; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008995:Monoamine Oxidase / Q000096:biosynthesis* / Q000302:isolation & purification; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D010627:Phenethylamines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012642:Selegiline / Q000494:pharmacology; D014439:Tyramine / Q000494:pharmacology
D008996:Monoamine Oxidase Inhibitors; D010627:Phenethylamines; D011994:Recombinant Proteins; D012642:Selegiline; C029261:phenethylamine; D008995:Monoamine Oxidase; D014439:Tyramine