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2013 | 60 | 4 |

Tytuł artykułu

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

Warianty tytułu

Języki publikacji

EN

Abstrakty

EN
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.

Słowa kluczowe

Wydawca

-

Rocznik

Tom

60

Numer

4

Opis fizyczny

p.585-590,fig.,ref.

Twórcy

autor
  • Departament of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland
autor
  • Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland
autor
  • Departament of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland

Bibliografia

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  • 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. 
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Typ dokumentu

Bibliografia

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