title: Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification.
abstract: Human pituitary glutaminyl cyclase (hQC) was expressed in Drosophila S2 cells under the control of an inducible metallothionene promoter and fused to the Drosophila immunoglobulin-binding protein signal sequence to enable secretion into the culture media. Expression levels reached 50 microg/mL culture media after 7 days of induction. The enzyme was purified to homogeneity directly from culture media by affinity chromatography on Reactive Blue 4-agarose using a step pH elution. The identity of the expressed protein was confirmed by peptide mass mapping and Western blotting. Glutaminyl cyclase was expressed as a fully active 37 kDa enzyme with kcat/Km values of 14.3, 9.3, and 2.4 mM(-1)s(-1) for the substrates Gln-Gln, Gln-NH(2), and Gln-t-butyl ester, respectively. The two cysteines were disulfide bonded, and the lone predicted glycosylation site, asparagine 49, was shown by both enzymatic deglycosylation of the expressed enzyme and site-directed mutagenesis to be glycosylated.
authors.affiliation: Department of Chemistry and Biochemistry, University of Southern Mississippi, P.O. Box 5043, Hattiesburg, MS 39406-5043, USA.
authors.forename: Rachell E
authors.initials: RE
authors.lastname: Booth
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Misquitta
authors.forename: Robert C
authors.initials: RC
authors.lastname: Bateman
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004220:Disulfides / Q000378:metabolism; D004330:Drosophila / Q000166:cytology* / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010902:Pituitary Gland / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013440:Sulfides / Q000378:metabolism
chemical_list: D004220:Disulfides; D011993:Recombinant Fusion Proteins; D013440:Sulfides; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase