Document Detail


BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies.
MedLine Citation:
PMID:  18782620     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in mammalian cells. Using these vectors, we show that the BacMam system in combination with the 293 GnTI(-) cell line can be used for production of milligram quantities of soluble glycoproteins. Moreover, for crystallization trials, the purified glycoproteins are sensitive to EndoH treatment resulting in a loss of the bulk of the attached N-linked glycosylation. In addition, we also show that a combination of the BacMam system and 293 GnTI(-) cell line can be used for producing milligram quantities of a GPCR-protein ligand complex suitable for crystallization trials.
Authors:
Abhiram Dukkipati; Hyun Ho Park; Deepa Waghray; Suzanne Fischer; K Christopher Garcia
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-08-22
Journal Detail:
Title:  Protein expression and purification     Volume:  62     ISSN:  1096-0279     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2008 Dec 
Date Detail:
Created Date:  2008-10-28     Completed Date:  2008-12-08     Revised Date:  2013-06-05    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  160-70     Citation Subset:  IM    
Affiliation:
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Baculoviridae / metabolism*
Base Sequence
Cell Line
Chemokine CX3CL1 / isolation & purification,  metabolism
Crystallography, X-Ray
Frizzled Receptors / chemistry,  isolation & purification,  metabolism
Genetic Vectors / genetics
Humans
Membrane Glycoproteins / chemistry*,  metabolism*
Mice
Molecular Sequence Data
Protein Structure, Tertiary
Receptor, Parathyroid Hormone, Type 1 / chemistry,  isolation & purification,  metabolism
Receptors, Chemokine / isolation & purification,  metabolism
Receptors, G-Protein-Coupled / chemistry,  isolation & purification,  metabolism
Recombinant Proteins / chemistry*,  metabolism*
Solubility
Transduction, Genetic / methods*
Transfection
Viral Proteins / isolation & purification,  metabolism
Grant Support
ID/Acronym/Agency:
//Howard Hughes Medical Institute
Chemical
Reg. No./Substance:
0/Chemokine CX3CL1; 0/Frizzled Receptors; 0/Fzd4 protein, mouse; 0/Membrane Glycoproteins; 0/Receptor, Parathyroid Hormone, Type 1; 0/Receptors, Chemokine; 0/Receptors, G-Protein-Coupled; 0/Recombinant Proteins; 0/US28 receptor, Cytomegalovirus; 0/Viral Proteins
Comments/Corrections

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