Human Pituitary Glutaminyl Cyclase: Expression in Insect Cells and Dye Affinity Purification

Authors

Rachell E. Booth, University of Southern Mississippi
Stephanie A. Misquitta, University of Southern Mississippi
Robert C. Bateman Jr., University of Southern MississippiFollow

Document Type

Article

Publication Date

11-1-2003

Department

Chemistry and Biochemistry

School

Mathematics and Natural Sciences

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 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–NH₂, 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.

Publication Title

Protein Expression and Purification

Volume

32

Issue

1

First Page

141

Last Page

146

Recommended Citation

Booth, R. E., Misquitta, S. A., Bateman, R. C. (2003). Human Pituitary Glutaminyl Cyclase: Expression in Insect Cells and Dye Affinity Purification. Protein Expression and Purification, 32(1), 141-146.
Available at: https://aquila.usm.edu/fac_pubs/9103