Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.
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 [(15)N]NH(4)Cl and [(13)C]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 (13)C,(15)N-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 (1)H-(13)C HSQC spectrum confirms (13)C 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.
Department of Chemistry, Macquarie University, Sydney, New South Wales, 2109, Australia.
L
L
Cubeddu
C X
CX
Moss
J D
JD
Swarbrick
A A
AA
Gooley
K L
KL
Williams
P M
PM
Curmi
M B
MB
Slade
B C
BC
Mabbutt
D000818:Animals; D000953:Antigens, Protozoan; D000954:Antigens, Surface / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D002250:Carbon Radioisotopes; D002852:Chromatography, Ion Exchange; D004023:Dictyostelium / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D009590:Nitrogen Radioisotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D002250:Carbon Radioisotopes; D008562:Membrane Glycoproteins; D009590:Nitrogen Radioisotopes; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C074496:prespore-specific antigen, Dictyostelium discoideum