Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification

• Published in: Protein expression and purification Vol. 32; no. 1; pp. 141 - 146
• Main Authors: Booth, Rachell E, Misquitta, Stephanie A, Bateman, Robert C
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2003
• Subjects: Amino Acid Sequence; Aminoacyltransferases - chemistry; Aminoacyltransferases - genetics; Aminoacyltransferases - isolation & purification; Animals; Cell Line; Chromatography, Affinity; Disulfides - metabolism; Drosophila - cytology; Drosophila - genetics; Glycosylation; Humans; Kinetics; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Pituitary Gland - enzymology; Protein Processing, Post-Translational; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Sulfides - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/S1046-5928(03)00226-2

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 μg/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 k cat/ K m 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.