Dictyostelium discoideum as expression host

Isotopic labeling of a recombinant glycoprotein for NMR studies

Liza Cubeddu*, Catherine X. Moss, James D. Swarbrick, Andrew A. Gooley, Keith L. Williams, Paul M G Curmi, Martin B. Slade, Bridget C. Mabbutt

*Corresponding author for this work

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [15N]NH4Cl and [13C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly 13C, 15N-labeled protein secreted by approximately 1010 D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional 1H-13C HSQC spectrum confirms 13C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)335-342
Number of pages8
JournalProtein Expression and Purification
Volume19
Issue number3
DOIs
Publication statusPublished - 2000

Fingerprint Dive into the research topics of 'Dictyostelium discoideum as expression host: Isotopic labeling of a recombinant glycoprotein for NMR studies'. Together they form a unique fingerprint.

Cite this