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

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.

SUBMITTER: Dukkipati A

PROVIDER: S-EPMC2637115 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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

Dukkipati Abhiram A Park Hyun Ho HH Waghray Deepa D Fischer Suzanne S Garcia K Christopher KC

Protein expression and purification 20080822 2

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 glyc ...[more]

PMID: 18782620

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