Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2000-9-6
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
1046-5928
pubmed:author
pubmed:copyrightInfo
Copyright 2000 Academic Press.
pubmed:issnType
Print
pubmed:volume
19
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
335-42
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:10910722-Animals, pubmed-meshheading:10910722-Antigens, Protozoan, pubmed-meshheading:10910722-Antigens, Surface, pubmed-meshheading:10910722-Carbon Radioisotopes, pubmed-meshheading:10910722-Chromatography, Ion Exchange, pubmed-meshheading:10910722-Dictyostelium, pubmed-meshheading:10910722-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:10910722-Escherichia coli, pubmed-meshheading:10910722-Gene Expression, pubmed-meshheading:10910722-Isotope Labeling, pubmed-meshheading:10910722-Mass Spectrometry, pubmed-meshheading:10910722-Membrane Glycoproteins, pubmed-meshheading:10910722-Nitrogen Radioisotopes, pubmed-meshheading:10910722-Nuclear Magnetic Resonance, Biomolecular, pubmed-meshheading:10910722-Protein Conformation, pubmed-meshheading:10910722-Protozoan Proteins, pubmed-meshheading:10910722-Recombinant Fusion Proteins
pubmed:year
2000
pubmed:articleTitle
Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.
pubmed:affiliation
Department of Chemistry, Macquarie University, Sydney, New South Wales, 2109, Australia.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't