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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Baculoviridae / metabolism*
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Base Sequence
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Cell Line
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Chemokine CX3CL1 / isolation & purification
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Chemokine CX3CL1 / metabolism
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Crystallography, X-Ray
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Frizzled Receptors / chemistry
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Frizzled Receptors / isolation & purification
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Frizzled Receptors / metabolism
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Genetic Vectors / genetics
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Humans
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / metabolism*
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Mice
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Molecular Sequence Data
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Protein Structure, Tertiary
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Receptor, Parathyroid Hormone, Type 1 / chemistry
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Receptor, Parathyroid Hormone, Type 1 / isolation & purification
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Receptor, Parathyroid Hormone, Type 1 / metabolism
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Receptors, Chemokine / isolation & purification
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Receptors, Chemokine / metabolism
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Receptors, G-Protein-Coupled / chemistry
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Receptors, G-Protein-Coupled / isolation & purification
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Receptors, G-Protein-Coupled / metabolism
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Recombinant Proteins / chemistry*
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Recombinant Proteins / metabolism*
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Solubility
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Transduction, Genetic / methods*
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Transfection
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Viral Proteins / isolation & purification
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Viral Proteins / metabolism
Substances
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Chemokine CX3CL1
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Frizzled Receptors
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Fzd4 protein, mouse
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Membrane Glycoproteins
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Receptor, Parathyroid Hormone, Type 1
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Receptors, Chemokine
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Receptors, G-Protein-Coupled
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Recombinant Proteins
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US28 receptor, Cytomegalovirus
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Viral Proteins