Protein stabilization through phage display

FEBS Letters

Chakravarty, Suvobrata²  Varadarajan, Raghavan²  Dübel, Stefan¹  Surolia, Avadhesha²  Mitra, Nivedita²  Queitsch, Iris¹ 

[1] University of Heidelberg, Molekulare Genetik, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany;Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India

关键词: Phage display;    Fragment complementation;    Thermal stability;    Epitope mapping;    RNase A;    bovine pancreatic ribonuclease;    S20;    residues 1–20 of RNase A;    S15;    residues 1–15 of S20;    S protein;    residues 21–124 of RNase A;    RNase S;    complex of S protein+S20 peptide;    S15p;    phage peptide selected from the phage display library;    ELISA;    enzyme-linked immunosorbent assay;    PBS;    phosphate-buffered saline;    cCMP;    cyclic cytidine monophosphate;    CD;    circular dichroism;    ITC;    isothermal titration calorimetry;    ΔH°;    change in molar enthalpy due to complex formation;    ΔG°;    change in molar free energy due to complex formation;    ΔC p;    change in molar heat capacity;    T m;    the temperature at which fraction of the peptide:protein complex is half of the total protein concentration;   

DOI  :  10.1016/S0014-5793(00)01725-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

RNase S consists of two proteolytic fragments of RNase A, residues 1–20 (S20) and residues 21–124 (S pro). A 15-mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25°C. However, the magnitudes of ΔH° and ΔC _p are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T _m of the S15p complex was found to be 10°C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.

【 授权许可】

Unknown