BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies.
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.
Dukkipati
Abhiram
A
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
Park
Hyun Ho
HH
Waghray
Deepa
D
Fischer
Suzanne
S
Garcia
K Christopher
KC
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D054428:Chemokine CX3CL1 / Q000302:isolation & purification / Q000378:metabolism; D018360:Crystallography, X-Ray; D051157:Frizzled Receptors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019707:Receptors, Chemokine / Q000302:isolation & purification / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism
D054428:Chemokine CX3CL1; D051157:Frizzled Receptors; C498581:Fzd4 protein, mouse; D008562:Membrane Glycoproteins; D044168:Receptor, Parathyroid Hormone, Type 1; D019707:Receptors, Chemokine; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C411191:US28 receptor, Cytomegalovirus; D014764:Viral Proteins
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