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2013 | 60 | 4 | 585-590
Article title

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

Content
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EN
Abstracts
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.
Year
Volume
60
Issue
4
Pages
585-590
Physical description
Dates
published
2013
received
2013-05-27
revised
2013-10-12
accepted
2013-11-12
(unknown)
2013-12-17
References
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Document Type
Publication order reference
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv60p585kz
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