Preferences help
enabled [disable] Abstract
Number of results
2013 | 60 | 4 | 585-590
Article title

Design, expression and characterization of a highly stable tetratricopeptide-based protein scaffold for phage display application

Title variants
Languages of publication
Tetratricopeptide repeat (TPR) is a structural motif mediating variety of protein-protein interactions. It has a high potential to serve as a small, stable and robust, non-immunoglobulin ligand binding scaffold. In this study, we showed the consensus approach to design the novel protein called designed tetratricopeptide repeat (dTPR), composed of three repeated 34 amino-acid tetratricopeptide motifs. The designed sequence was efficiently overexpressed in E. coli and purified to homogeneity. Recombinant dTPR is monomeric in solution and preserves its secondary structure within the pH range from 2.0 to 11.0. Its denaturation temperature at pH 7.5 is extremely high (104.5°C) as determined by differential scanning calorimetry. At extreme pH values the protein is still very stable: denaturation temperature is 90.1°C at pH 2.0 and 60.4°C at pH 11. Chemical unfolding of the dTPR is a cooperative, two-state process both at pH 7.5 and 2.0. The free energy of denaturation in the absence of denaturant equals to 15.0 kcal/mol and 13.5 kcal/mol at pH 7.5 and 2.0, respectively. Efficient expression and extraordinary biophysical properties make dTPR a promising framework for a biotechnological application, such as generation of specific ligand- binding molecules.
Physical description
  • Departament of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Departament of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Alfarano P, Varadamsetty G, Ewald C, Parmeggiani F, Pellarin R, Zerbe O, Plückthun A, Caflisch A (2012) Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy. Protein Sci 21: 1298-1314.
  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.
  • Becktel WJ, Schellman JA (1987) Protein stability curves. Biopolymers 26: 1859-1877.
  • Binz HK, Amstutz P, Plückthun A (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat Biotechnol 23: 1257-1268.
  • Binz HK, Stumpp MT, Forrer P, Amstutz P, Plückthun A (2003) Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J Mol Biol 332: 489-503.
  • Cortajarena AL, Kajander T, Pan W, Cocco MJ, Regan L (2004) Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins. Protein Eng Des Sel 17: 399-409.
  • Cortajarena AL, Regan L (2006) Ligand binding by TPR domains. Protein Sci 15: 1193-1198.
  • Cortajarena AL, Yi F, Regan L (2008) Designed TPR modules as novel anticancer agents. ACS Chem Biol 3: 161-166.
  • D'Andrea LD, Regan L (2003) TPR proteins: the versatile helix. Trends Biochem Sci 28: 655-662.
  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2. Bioinformatics 23: 2947-2948.
  • Lipovsek D (2011) Adnectins: engineered target-binding protein therapeutics. Protein Eng Des Sel 24: 3-9.
  • Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY (2010) Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett 584: 2670-2680.
  • Löfblom J, Frejd FY, Ståhl S (2011) Non-immunoglobulin based protein scaffolds. Curr Opin Biotechnol 22: 843-838.
  • Main ER, Xiong Y, Cocco MJ, D'Andrea L, Regan L (2003) Design of stable alpha-helical arrays from an idealized TPR motif. Structure 11: 497-508.
  • Makhatadze GI, Privalov PL (1995) Energetics of protein structure. Adv Protein Chem 47: 307-425.
  • Markert Y, Köditz J, Mansfeld J, Arnold U, Ulbrich-Hofmann R (2001) Increased proteolytic resistance of ribonuclease A by protein engineering. Protein Eng 14: 791-796.
  • Perez-Iratxeta C, Andrade-Navarro MA (2008) K2D2: estimation of protein secondary structure from circular dichroism spectra. BMC Struct Biol 8: 25.
  • Privalov PL (1979) Stability of proteins: small globular proteins. Adv Protein Chem 33: 167-241.
  • Schultz J, Milpetz F, Bork P, Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA 95: 5857-5864.
  • Sikorski RS, Boguski MS, Goebl M, Hieter P (1990) A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell 60: 307-317.
  • Skerra A (2003) Imitating the humoral immune response. Curr Opin Chem Biol 7: 683-693.
  • Skerra A (2007) Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol 18: 295-304.
  • Skerra A (2008) Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. FEBS J 275: 2677-2683.
  • Steipe B, Schiller B, Pluckthun A, Steinbacher S (1994) Sequence statistics reliably predict stabilizing mutations in a protein domain. J Mol Biol 240: 188-192.
  • Varadamsetty G, Tremmel D, Hansen S, Parmeggiani F, Plückthun A (2012) Designed armadillo repeat proteins: library generation, characterization and selection of peptide binders with high specificity. J Mol Biol 424: 68-87.
  • Wetzel SK, Settanni G, Kenig M, Binz HK, Plückthun A (2008) Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins. J Mol Biol 376: 241-257.
  • Wezner-Ptasińska M, Krowarsch D, Otlewski J (2011) Design and characteristics of a stable protein scaffold for specific binding based on variable lymphocyte receptor sequences. Biochim Biophys Acta 1814: 1140-1145.
  • Zahnd E, Wyler JM, Schwenk D, Steiner MC, Lawrence NM, McKern F, Pecorari CW, Ward TO, Joos A, Plückthun A (2007) Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2. J Mol Biol 369: 1015-1028.
  • Zakrzewska M, Krowarsch D, Wiedlocha A, Olsnes S, Otlewski J (2005) Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. J Mol Biol 352: 860-875.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.