Two major strategies are currently used for stabilizing proteins: in vitro evolution and computational design. Here, we used gene libraries of the β1 domain of the streptococcal protein G (Gβ1) and Proside, an in vitro selection method, to identify stabilized variants of this protein. In the Gβ1 libraries, the codons for the four boundary positions 16, 18, 25, and 29 were randomized. Many Gβ1 variants with strongly increased thermal stabilities were found in 11 selections performed with five independent libraries. Previously, Mayo and co-workers used computational design to stabilize Gβ1 by sequence optimization at the same positions. Their best variant ranked third within the panel of the selected variants. None of the ten computed sequences was found in the Proside selections, because several computed residues for positions 18 and 29 were not optimal for stability.
Submitter: Marie Ary
Submission Date: Feb. 20, 2017, 12:56 p.m.
|Number of data points||219|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: [GdmCl]m ; Experimental Assay: ΔHD(Tm) ; Experimental Assay: ΔGD (70°C) ; Experimental Assay: ΔGD (30°C) ; Experimental Assay: Tm in 2.5M GdmCl pH7.0 ; Derived Quantity: ΔΔGD(70°C)|
|Libraries||GdmCl unfolding of selected strongly stabilized GB1 variants (Table 3) ; in vitro selection of 4 boundary positions compared to CPD variants (Table 2)|