Document Detail


An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells.
MedLine Citation:
PMID:  17063379     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells. A key aspect of this work was to reduce the amount of glutamine in the cell culture medium while maintaining sufficient energy yielding metabolites for vigorous growth by supplementing with glucose and algae-derived amino acids. We present an analysis of cell growth and protein production in Sf9 insect cells expressing secreted Thy1-GFP fusion construct. We also demonstrate isotopic enrichment of the Thy-1 protein backbone with 15N and carbohydrates with 13C by NMR spectroscopy.
Authors:
Wendy J Walton; Agnieszka J Kasprzak; Joan T Hare; Timothy M Logan
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2006-10-25
Journal Detail:
Title:  Journal of biomolecular NMR     Volume:  36     ISSN:  0925-2738     ISO Abbreviation:  J. Biomol. NMR     Publication Date:  2006 Dec 
Date Detail:
Created Date:  2006-11-22     Completed Date:  2007-02-27     Revised Date:  2007-12-03    
Medline Journal Info:
Nlm Unique ID:  9110829     Medline TA:  J Biomol NMR     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  225-33     Citation Subset:  IM    
Affiliation:
Institute of Molecular Biophysics, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / analysis
Animals
Antigens, Thy-1 / isolation & purification,  metabolism
Carbohydrates / analysis
Carbon Isotopes / chemistry*
Cell Line
Cell Survival
Culture Media / chemistry,  economics*
Glucose / analysis
Glutamine / pharmacology
Glycoproteins / metabolism*
Magnetic Resonance Spectroscopy / methods
Nitrogen Isotopes / chemistry*
Proteins / analysis
Spodoptera / cytology,  metabolism*
Grant Support
ID/Acronym/Agency:
GM66340/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Amino Acids; 0/Antigens, Thy-1; 0/Carbohydrates; 0/Carbon Isotopes; 0/Culture Media; 0/Glycoproteins; 0/Nitrogen Isotopes; 0/Proteins; 50-99-7/Glucose; 56-85-9/Glutamine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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