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2012 | 59 | 3 | 383-390
Article title

A novel, stable, helical scaffold as an alternative binder - construction of phage display libraries

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Specific, high affinity binding macromolecules are of great importance for biomedical and biotechnological applications. The most popular classical antibody-based molecules have recently been challenged by alternative scaffolds with desirable biophysical properties. Phage display technology applied to such scaffolds allows generation of potent affinity reagents by in vitro selection. Here, we report identification and characterization of a novel helical polypeptide with advantageous biophysical properties as a template for construction of phage display libraries. A three-helix bundle structure, based on Measles virus phosphoprotein P shows a very favourable stability and solubility profile. We designed, constructed and characterized six different types of phage display libraries based on the proposed template. Their functional size of over 109 independent clones, balanced codon bias and decent display level are key parameters attesting to the quality and utility of the libraries. The new libraries are a promising tool for isolation of high affinity binders based on a small helical scaffold which could become a convenient alternative to antibodies.
Physical description
  • Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Amstutz P, Forrer P, Zahnd C, Pluckthun A (2001) In vitro display technologies: novel developments and applications. Curr Opin Biotechnol 12: 400-405.
  • Barbas CF 3rd (1995) Synthetic human antibodies. Nat Med 1: 837-839.
  • Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter MG, Pluckthun A (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 22: 575-582.
  • Binz HK, Amstutz P, Pluckthun A (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat Biotechnol 23: 1257-1268.
  • Binz HK, Pluckthun A (2005) Engineered proteins as specific binding reagents. Curr Opin Biotechnol 16: 459-469.
  • Blanchard L, Tarbouriech N, Blackledge M, Timmins P, Burmeister WP, Ruigrok RW, Marion D (2004) Structure and dynamics of the nucleocapsid-binding domain of the Sendai virus phosphoprotein in solution. Virology 319: 201-211.
  • Emberley ED, Murphy LC, Watson PH (2004) S100A7 and the progression of breast cancer. Breast Cancer Res 6: 153-159.
  • Fellouse FA, Barthelemy PA, Kelley RF, Sidhu SS (2006) Tyrosine plays a dominant functional role in the paratope of a synthetic antibody derived from a four amino acid code. J Mol Biol 357: 100-114.
  • Fellouse FA, Wiesmann C, Sidhu SS (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci USA 101: 12467-12472.
  • Friedman M, Orlova A, Johansson E, Eriksson TL, Hoiden-Guthenberg I, Tolmachev V, Nilsson FY, Stahl S (2008) Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. J Mol Biol 376: 1388-1402.
  • Kesting MR, Sudhoff H, Hasler RJ, Nieberler M, Pautke C, Wolff KD, Wagenpfeil S, Al-Benna S, Jacobsen F, Steinstraesser L (2009) Psoriasin (S100A7) up-regulation in oral squamous cell carcinoma and its relation to clinicopathologic features. Oral Oncology 45: 731-736.
  • Kingston RL, Gay LS, Baase WS, Matthews BW (2008) Structure of the nucleocapsid-binding domain from the mumps virus polymerase; an example of protein folding induced by crystallization. J Mol Biol 379: 719-731.
  • Koide A, Bailey CW, Huang X, Koide S (1998) The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol 284: 1141-1151.
  • Koide A, Gilbreth RN, Esaki K, Tereshko V, Koide S (2007) High-affinity single-domain binding proteins with a binary-code interface. Proc Natl Acad Sci USA 104: 6632-6637.
  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
  • Lendel C, Dogan J, Hard T (2006) Structural basis for molecular recognition in an affibody:affibody complex. J Mol Biol 359: 1293-1304.
  • Ling MM (2003) Large antibody display libraries for isolation of high-affinity antibodies. Combinatorial Chemistry & High Throughput Screening 6: 421-432.
  • Longhi S, Receveur-Brechot V, Karlin D, Johansson K, Darbon H, Bhella D, Yeo R, Finet S, Canard B (2003) The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein. J Biol Chem 278: 18638-18648.
  • Lucast LJ, Batey RT, Doudna JA (2001) Large-scale purification of a stable form of recombinant tobacco etch virus protease. BioTechniques 30: 544-546 548-550 passim.
  • Milovnik P, Ferrari D, Sarkar CA, Pluckthun A (2009) Selection and characterization of DARPins specific for the neurotensin receptor 1. Protein Engineering Design & Selection 22: 357-366.
  • Morgan MR, Jazayeri M, Ramsay AG, Thomas GJ, Boulanger MJ, Hart IR, Marshall JF (2011) Psoriasin (S100A7) associates with integrin beta6 subunit and is required for alphavbeta6-dependent carcinoma cell invasion. Oncogene 30: 1422-1435.
  • Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA (1997) Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nat Biotechnol 15: 772-777.
  • Nygren PA (2008) Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 275: 2668-2676.
  • Nygren PA, Uhlen M (1997) Scaffolds for engineering novel binding sites in proteins. Curr Opin Struct Biol 7: 463-469.
  • Orlova A, Magnusson M, Eriksson TL, Nilsson M, Larsson B, Hoiden-Guthenberg I, Widstrom C, Carlsson J, Tolmachev V, Stahl S, Nilsson FY (2006) Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res 66: 4339-4348.
  • Russel MLH, Clackson T (2004) Introduction to phage biology and phage display. In Phage Display: A Practical Approach. Oxford university Press.
  • Sidhu SS, Kossiakoff AA (2007) Exploring and designing protein function with restricted diversity. Curr Opin Chem Biol 11: 347-354.
  • Skerra A (2007) Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol 18: 295-304.
  • Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315-1317.
  • Smith GP, Scott JK (1993) Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol 217: 228-257.
  • Steiner D, Forrer P, Pluckthun A (2008) Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. J Mol Biol 382: 1211-1227.
  • Steiner D, Forrer P, Stumpp MT, Pluckthun A (2006) Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nat Biotechnol 24: 823-831.
  • Tamaskovic R, Simon M, Stefan N, Schwill M, Pluckthun A (2012) Designed ankyrin repeat proteins (DARPins) from research to therapy. Methods Enzymol 503: 101-134.
  • Wahlberg E, Lendel C, Helgstrand M, Allard P, Dincbas-Renqvist V, Hedqvist A, Berglund H, Nygren PA, Hard T (2003) An affibody in complex with a target protein: structure and coupled folding. Proc Natl Acad Sci USA 100: 3185-3190.
  • Wikman M, Steffen AC, Gunneriusson E, Tolmachev V, Adams GP, Carlsson J, Stahl S (2004) Selection and characterization of HER2/neu-binding affibody ligands. Protein Eng Des Sel 17: 455-462.
  • Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, Jones RB, Koide A, Superti-Furga G, Koide S (2010) A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain. Nat Struct Mol Biol 17: 519-527.
  • Zahnd C, Pecorari F, Straumann N, Wyler E, Pluckthun A (2006) Selection and characterization of Her2 binding-designed ankyrin repeat proteins. J Biol Chem 281: 35167-35175.
  • Zahnd C, Wyler E, Schwenk JM, Steiner D, Lawrence MC, McKern NM, Pecorari F, Ward CW, Joos TO, Pluckthun A (2007) A designed ankyrin repeat protein evolved to picomolar affinity to Her2. J Mol Biol 369: 1015-1028.
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