期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 363, 期 2, 页码 545-557出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2006.08.034
关键词
protein stability; in-vitro selection; phage display; error-prone PCR; computational protein design
An in-vitro selection strategy was used to obtain strongly stabilized variants of the 131 domain of protein G (G beta 1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in G l libraries that were created by error-prone PCR, and then, after randomization of these positions by saturation mutagenesis, strongly stabilized variants were selected. For both steps the in-vitro selection method Proside was employed. Proside links the stability of a protein with the infectivity of a filamentous phage. Ultimately, residues from the two best selected variants were combined in a single G l molecule. This variant with the four mutations E15V, T16L, T181, and N37L showed an increase of 35.1 degrees C in the transition midpoint and of 28.5 kJ mol(-1) (at 70 degrees C) in the Gibbs free energy of stabilization. It was considerably more stable than the best variant from a previous Proside selection, in which positions were randomized that had originally been identified by computational design. Only a single substitution (T181) was found in both selections. The best variants from the present selection showed a higher cooperativity of thermal unfolding, as indicated by an increase in the enthalpy of unfolding by about 60 kJ mol(-1). This increase is apparently correlated with the presence of Leu residues that were selected at the positions 16 and 37. (c) 2006 Elsevier Ltd. All rights reserved.
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