Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification.
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.
Booth
Rachell E
RE
Department of Chemistry and Biochemistry, University of Southern Mississippi, P.O. Box 5043, Hattiesburg, MS 39406-5043, USA.
Misquitta
Stephanie A
SA
Bateman
Robert C
RC
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
D004220:Disulfides; D011993:Recombinant Fusion Proteins; D013440:Sulfides; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase