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PMID: 18782620 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Protein expression and purification ·Vol. 62 ·No. 2 ·2008-12-00 ·Pages 160-70

Dukkipati A, Park HH, Waghray D, Fischer S, Garcia KC

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.

MeSH Terms
Animals Baculoviridae/metabolism Base Sequence Cell Line Chemokine CX3CL1/isolation & purification,metabolism Crystallography, X-Ray Frizzled Receptors/chemistry,isolation & purification,metabolism Genetic Vectors/genetics Humans Membrane Glycoproteins/chemistry,metabolism Mice Molecular Sequence Data Protein Structure, Tertiary Receptor, Parathyroid Hormone, Type 1/chemistry,isolation & purification,metabolism Receptors, Chemokine/isolation & purification,metabolism Receptors, G-Protein-Coupled/chemistry,isolation & purification,metabolism Recombinant Proteins/chemistry,metabolism Solubility Transduction, Genetic/methods Transfection Viral Proteins/isolation & purification,metabolism
Chemicals
Chemokine CX3CL1 Frizzled Receptors Fzd4 protein, mouse Membrane Glycoproteins Receptor, Parathyroid Hormone, Type 1 Receptors, Chemokine Receptors, G-Protein-Coupled Recombinant Proteins US28 receptor, Cytomegalovirus Viral Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Dukkipati Abhiram
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
Park Hyun Ho
Waghray Deepa
Fischer Suzanne
Garcia K Christopher
References (30)
30 references, click to expand
  1. In vitro reconstitution and preparative purification of complexes between the chemokine receptor CXCR4 and its ligands SDF-1alpha, gp120-CD4 and AMD3100.
    Protein Expr Purif. 2006 Dec;50(2):203-14 PMID: 16962791
  2. Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by agonist-independent phosphorylation.
    J Biol Chem. 2002 Nov 22;277(47):45122-8 PMID: 12244063
  3. Comparison of intron-dependent and intron-independent gene expression.
    Mol Cell Biol. 1988 Oct;8(10):4395-405 PMID: 3185553
  4. Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28.
    FEBS Lett. 1998 Dec 18;441(2):209-14 PMID: 9883886
  5. Implementation of BacMam virus gene delivery technology in a drug discovery setting.
    Drug Discov Today. 2007 May;12(9-10):396-403 PMID: 17467576
  6. Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.
    J Biol Chem. 2001 Sep 7;276(36):33906-14 PMID: 11432858
  7. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.
    J Virol. 1999 Apr;73(4):2886-92 PMID: 10074136
  8. Elution of antibodies from a Protein-A column by aqueous arginine solutions.
    Protein Expr Purif. 2004 Aug;36(2):244-8 PMID: 15249046
  9. Parathyroid hormone: past and present.
    J Endocrinol. 2005 Dec;187(3):311-25 PMID: 16423810
  10. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector.
    Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):127-32 PMID: 9874783
  11. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy.
    Nat Genet. 2002 Oct;32(2):326-30 PMID: 12172548
  12. Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors.
    J Gen Virol. 1997 Oct;78 ( Pt 10):2657-64 PMID: 9349488
  13. A new class of membrane-bound chemokine with a CX3C motif.
    Nature. 1997 Feb 13;385(6617):640-4 PMID: 9024663
  14. Purification and biochemical characterization of the D6 chemokine receptor.
    Biochem J. 2004 Apr 15;379(Pt 2):263-72 PMID: 14723600
  15. The N-terminal fragment of human parathyroid hormone receptor 1 constitutes a hormone binding domain and reveals a distinct disulfide pattern.
    Biochemistry. 2000 Aug 1;39(30):8878-87 PMID: 10913300
  16. Human cytomegalovirus open reading frame US28 encodes a functional beta chemokine receptor.
    J Biol Chem. 1994 Nov 18;269(46):28539-42 PMID: 7961796
  17. Large-scale transient expression of therapeutic proteins in mammalian cells.
    Methods Mol Biol. 2005;308:87-98 PMID: 16082028
  18. Orbital shaker technology for the cultivation of mammalian cells in suspension.
    Biotechnol Bioeng. 2005 Feb 20;89(4):400-6 PMID: 15619325
  19. Recombinant expression and purification of the N-terminal extracellular domain of the parathyroid hormone receptor.
    Protein Expr Purif. 2007 Jul;54(1):87-93 PMID: 17448676
  20. Efficient expression of secreted proteases via recombinant BacMam virus.
    Protein Expr Purif. 2007 Mar;52(1):104-16 PMID: 17129735
  21. Efficient gene transfer into human hepatocytes by baculovirus vectors.
    Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10099-103 PMID: 7479733
  22. Baculovirus-mediated gene transfer into mammalian cells.
    Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2348-52 PMID: 8637876
  23. Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor.
    Biochemistry. 1996 Dec 10;35(49):15890-5 PMID: 8961954
  24. Identification of a chemokine receptor encoded by human cytomegalovirus as a cofactor for HIV-1 entry.
    Science. 1997 Jun 20;276(5320):1874-8 PMID: 9188536
  25. Sialylated O-glycans and sulfated tyrosines in the NH2-terminal domain of CC chemokine receptor 5 contribute to high affinity binding of chemokines.
    J Exp Med. 2001 Dec 3;194(11):1661-73 PMID: 11733580
  26. The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry.
    J Biol Chem. 2002 Aug 16;277(33):29484-9 PMID: 12034737
  27. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line.
    Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13419-24 PMID: 12370423
  28. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.
    Nucleic Acids Res. 1990 Feb 25;18(4):937-47 PMID: 1690394
  29. The cytomegalovirus US28 protein binds multiple CC chemokines with high affinity.
    Biochem Biophys Res Commun. 1995 Jun 6;211(1):325-30 PMID: 7540006
  30. The Frizzled family: receptors for multiple signal transduction pathways.
    Genome Biol. 2004;5(7):234 PMID: 15239825
Article Info
Journal
Protein expression and purification
Abbr.
Protein Expr Purif
ISSN
1096-0279
DOI
10.1016/j.pep.2008.08.004
Published
2008-12-00
Epub
2008-00-22
Pages
160-70
Language
English
Region
United States
NLM ID
9101496
PMCID
PMC2637115
Subset
IM
Grants
Howard Hughes Medical Institute · United States
External Links
PubMed source DOI: 10.1016/j.pep.2008.08.004 PMC Full Text View journal details
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