title: Affilin molecules selected against the human papillomavirus E7 protein inhibit the proliferation of target cells.
abstract: Intracellular binding proteins can be applied as research tools for target validation and study of protein function in cells and potentially as therapeutics. The success of intracellular binding reagents depends on their affinity and specificity for target molecules, although their stability and functionality in the intracellular environment actually determine their usefulness for such application. Alternative binding proteins derived from scaffolds devoid of disulfide bonds are well suited for intracellular use, as their folding and stability are usually not impaired under reducing conditions. Here, we describe the generation of intracellular binding reagents called Affilin, based on the human gammaB-crystallin scaffold. The target was human papillomavirus E7 protein implicated in the development of cervical cancer. E7 binders were selected from the combinatorial gammaB-crystallin library by conventional phage display technique. Affilin variants specifically bound the E7 protein with affinities in the nanomolar range. Intracellular expression of Affilin molecules in E7-positive cells led to inhibition of cellular proliferation. The effect was specific, as the growth of E7-negative cells or cells expressing the wild-type gammaB-crystallin scaffold remained unaffected. These results demonstrate that the gammaB-crystallin scaffold allows the de novo generation of alternative binding proteins, which are suitable for intracellular applications as they retain their functionality in the reducing environment of mammalian cells.
authors.affiliation: Institute of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
authors.forename: Ewa A
authors.initials: EA
authors.lastname: Mirecka
authors.forename: Thomas
authors.initials: T
authors.lastname: Hey
authors.forename: Ulrike
authors.initials: U
authors.lastname: Fiedler
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Mechthild
authors.initials: M
authors.lastname: Hatzfeld
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation; D005260:Female; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D050725:Papillomavirus E7 Proteins / Q000737:chemistry*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D038222:gamma-Crystallins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D050725:Papillomavirus E7 Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; C107738:crystallin, gammaB; D038222:gamma-Crystallins