| Autor: |
Booth RE; Department of Chemistry and Biochemistry, University of Southern Mississippi, P.O. Box 5043, Hattiesburg, MS 39406-5043, USA., Misquitta SA, Bateman RC Jr |
| Jazyk: |
angličtina |
| Zdroj: |
Protein expression and purification [Protein Expr Purif] 2003 Nov; Vol. 32 (1), pp. 141-6. |
| DOI: |
10.1016/S1046-5928(03)00226-2 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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