Dictyostelium discoideum as expression host: Isotopic labeling of a recombinant glycoprotein for NMR studies

摘要

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 [N-15]NH4Cl and [C-13]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 C-13, N-15-labeled protein secreted by approximately 10(10) 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 H-1-C-13 HSQC spectrum confirms C-13 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. [References: 26]