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Protein Expr Purif. 2008 Dec;62(2):160-70. doi: 10.1016/j.pep.2008.08.004. Epub 2008 Aug 22.

BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies.

Author information

1
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.

Abstract

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.

PMID:
18782620
PMCID:
PMC2637115
DOI:
10.1016/j.pep.2008.08.004
[Indexed for MEDLINE]
Free PMC Article

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