title: Molecular cloning, overexpression, and an efficient one-step purification of α5β1 integrin.
abstract: The α5β1 integrin heterodimer is involved in many cellular processes and is an anti-cancer therapeutic target. Therefore, access to quantities of protein suitable for studies aimed at understanding its biological functions is important. To this end, a large-scale protein expression system, utilizing the recombinant baculovirus/SF9 insect cell expression system, was created to produce the extracellular domain of the α5β1 integrin. An incorporated 8X-histidine tag enabled one-step nickel-column purification. Following sequence confirmation by LC-MS/MS, the conformation of the heterodimer was characterized by native dot blot and negative stain electron microscopy. Cellular transduction inhibition studies confirmed biological activity. The system allows expression and purification of α5β1 integrin in quantities suitable for an array of different experiments including structural biology.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Tartaglia
authors.forename: Antonette
authors.initials: A
authors.lastname: Bennett
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Plattner
authors.forename: Nicholas
authors.initials: N
authors.lastname: Muzyczka
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002853:Chromatography, Liquid; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D007313:Insecta; D039081:Integrin alpha5beta1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D015854:Up-Regulation
chemical_list: D039081:Integrin alpha5beta1; D011994:Recombinant Proteins
keywords: Electron microscopy; Recombinant baculovirus; pFastBac1 vector; α5β1 Integrin expression