title: Construction, expression, and purification of recombinant αVβ5 integrin.
abstract: A recombinant integrin expression system has been created for the large-scale production of αVβ5 integrin extracellular domains that take advantage of Fos and Jun dimerization for expression in bacterial, insect, and mammalian cells. This utilizes an all-in-one vector, pQE-TriSystem, with molecular machinery for parallel expression without the need of additional subcloning. Optimal expression in HEK293 cells was determined by a time course analysis. The heterodimer was purified in a one-step nickel column purification scheme, and the sequence and functional state were confirmed by mass spectrometry and inhibition assays, respectively. The yields of αVβ5 integrin obtained are in quantities suitable for multiple applications including structural biology and functional assays.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, United States.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Tartaglia
authors.forename: Antonette
authors.initials: A
authors.lastname: Bennett
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Woodhouse
authors.forename: Fikret
authors.initials: F
authors.lastname: Aydemir
authors.forename: Nicholas
authors.initials: N
authors.lastname: Muzyczka
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D019030:Receptors, Vitronectin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; C064660:integrin alphaVbeta5