title: 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.
authors.affiliation: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Abhiram
authors.initials: A
authors.lastname: Dukkipati
authors.forename: Hyun Ho
authors.initials: HH
authors.lastname: Park
authors.forename: Deepa
authors.initials: D
authors.lastname: Waghray
authors.forename: Suzanne
authors.initials: S
authors.lastname: Fischer
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
mesh_terms: 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
chemical_list: 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
references.citation: Protein Expr Purif. 2006 Dec;50(2):203-14
references.pmid: 16962791
references.citation: J Biol Chem. 2002 Nov 22;277(47):45122-8
references.pmid: 12244063
references.citation: Mol Cell Biol. 1988 Oct;8(10):4395-405
references.pmid: 3185553
references.citation: FEBS Lett. 1998 Dec 18;441(2):209-14
references.pmid: 9883886
references.citation: Drug Discov Today. 2007 May;12(9-10):396-403
references.pmid: 17467576
references.citation: J Biol Chem. 2001 Sep 7;276(36):33906-14
references.pmid: 11432858
references.citation: J Virol. 1999 Apr;73(4):2886-92
references.pmid: 10074136
references.citation: Protein Expr Purif. 2004 Aug;36(2):244-8
references.pmid: 15249046
references.citation: J Endocrinol. 2005 Dec;187(3):311-25
references.pmid: 16423810
references.citation: Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):127-32
references.pmid: 9874783
references.citation: Nat Genet. 2002 Oct;32(2):326-30
references.pmid: 12172548
references.citation: J Gen Virol. 1997 Oct;78 ( Pt 10):2657-64
references.pmid: 9349488
references.citation: Nature. 1997 Feb 13;385(6617):640-4
references.pmid: 9024663
references.citation: Biochem J. 2004 Apr 15;379(Pt 2):263-72
references.pmid: 14723600
references.citation: Biochemistry. 2000 Aug 1;39(30):8878-87
references.pmid: 10913300
references.citation: J Biol Chem. 1994 Nov 18;269(46):28539-42
references.pmid: 7961796
references.citation: Methods Mol Biol. 2005;308:87-98
references.pmid: 16082028
references.citation: Biotechnol Bioeng. 2005 Feb 20;89(4):400-6
references.pmid: 15619325
references.citation: Protein Expr Purif. 2007 Jul;54(1):87-93
references.pmid: 17448676
references.citation: Protein Expr Purif. 2007 Mar;52(1):104-16
references.pmid: 17129735
references.citation: Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10099-103
references.pmid: 7479733
references.citation: Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2348-52
references.pmid: 8637876
references.citation: Biochemistry. 1996 Dec 10;35(49):15890-5
references.pmid: 8961954
references.citation: Science. 1997 Jun 20;276(5320):1874-8
references.pmid: 9188536
references.citation: J Exp Med. 2001 Dec 3;194(11):1661-73
references.pmid: 11733580
references.citation: J Biol Chem. 2002 Aug 16;277(33):29484-9
references.pmid: 12034737
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13419-24
references.pmid: 12370423
references.citation: Nucleic Acids Res. 1990 Feb 25;18(4):937-47
references.pmid: 1690394
references.citation: Biochem Biophys Res Commun. 1995 Jun 6;211(1):325-30
references.pmid: 7540006
references.citation: Genome Biol. 2004;5(7):234
references.pmid: 15239825