title: High-level expression of a full-length Eph receptor.
abstract: Eph receptors are the largest family of Receptor Tyrosine Kinases containing a single membrane-spanning segment. They are involved in a various developmental and cell-cell communication events. Although there is extensive structural information available on both the extra- and intracellular regions of Eph's in isolation, no structures are available for the entire receptor. To facilitate structural studies on functionally relevant Eph/ephrin complexes, we have developed an expression system for producing the full-length human EphA2 receptor. We successfully expressed milligram amounts of the receptor using baculovirus-based vector and insect cells. We were also able to extract the protein from the cell membranes and purify it to near homogeneity in two simple steps. The purified receptor was shown to retain its biological activity in terms of both binding to its functional ligands and being able to auto-phosphorylate the key tyrosine residues of the cytoplasmic kinase domain.
authors.affiliation: Joint Biotechnology Lab, University of Turku, BioCity 6A, 20520 Turku, Finland.
authors.forename: Sari
authors.initials: S
authors.lastname: Paavilainen
authors.forename: David
authors.initials: D
authors.lastname: Grandy
authors.forename: Eveliina
authors.initials: E
authors.lastname: Karelehto
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Chang
authors.forename: Petri
authors.initials: P
authors.lastname: Susi
authors.forename: Hediye
authors.initials: H
authors.lastname: Erdjument-Bromage
authors.forename: Dimitar
authors.initials: D
authors.lastname: Nikolov
authors.forename: Juha
authors.initials: J
authors.lastname: Himanen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D010766:Phosphorylation; D036104:Receptor, EphA2 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D036104:Receptor, EphA2
keywords: Eph receptors; Insect cell; Overexpression; Receptor Tyrosine Kinases; Transmembrane proteins
references.citation: Neuron. 1996 Jul;17(1):9-19
references.pmid: 8755474
references.citation: Mol Cells. 2010 May;29(5):443-8
references.pmid: 20432069
references.citation: J Biol Chem. 1992 Feb 15;267(5):3262-7
references.pmid: 1737782
references.citation: Comb Chem High Throughput Screen. 2009 Sep;12(8):791-800
references.pmid: 19758119
references.citation: Cell Mol Life Sci. 2006 Jan;63(1):36-51
references.pmid: 16314922
references.citation: Nat Chem Biol. 2012 Jul 18;8(8):670-3
references.pmid: 22810761
references.citation: Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):10860-5
references.pmid: 20505120
references.citation: J Biol Chem. 2004 Mar 5;279(10):9522-31
references.pmid: 14660665
references.citation: Methods. 2002 Mar;26(3):260-9
references.pmid: 12054882
references.citation: Nature. 2009 May 21;459(7245):344-6
references.pmid: 19458709
references.citation: Nature. 2009 May 21;459(7245):356-63
references.pmid: 19458711
references.citation: Curr Opin Cell Biol. 2007 Apr;19(2):117-23
references.pmid: 17306972
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):9953-8
references.pmid: 18632560
references.citation: N Engl J Med. 2001 Apr 5;344(14):1031-7
references.pmid: 11287972
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):35-42
references.pmid: 22044883
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9773-8
references.pmid: 16785428
references.citation: Science. 2005 Dec 2;310(5753):1461-5
references.pmid: 16322449
references.citation: Chem Biol. 2011 Mar 25;18(3):361-71
references.pmid: 21439481
references.citation: Nature. 1996 Oct 24;383(6602):722-5
references.pmid: 8878483
references.citation: J Chromatogr A. 1998 Nov 27;826(2):167-81
references.pmid: 9871337
references.citation: Protein Sci. 1999 Apr;8(4):921-9
references.pmid: 10211839
references.citation: Curr Opin Cell Biol. 2009 Apr;21(2):140-6
references.pmid: 19269802
references.citation: Nature. 1998 Dec 3;396(6710):486-91
references.pmid: 9853759
references.citation: Protein Sci. 2008 Oct;17(10):1740-7
references.pmid: 18628240
references.citation: Handb Exp Pharmacol. 2009;(190):3-28
references.pmid: 19096770