Gu H, Yi Q, Bray S T, Riddle D S, Shiau A K, Baker D
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Protein Sci. 1995 Jun;4(6):1108-17. doi: 10.1002/pro.5560040609.
We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.
我们开发了一种噬菌体展示系统，该系统提供了一种从大型组合文库中筛选折叠的消化链球菌蛋白L的IgG结合域变体的方法。选择方案的基本前提是蛋白L与IgG的结合要求蛋白正确折叠。通过结合分子生物学和生物物理方法，我们证明了这一假设是正确的。首先，噬菌体选择程序强烈地筛选出蛋白L中破坏折叠但不在IgG结合界面的点突变。其次，从蛋白L前三分之一被随机化的文库中回收的变体能够正确折叠。所选群体中的序列变异程度惊人：在被诱变的14个残基中，变体有多达9个取代。该方法提供了一种对“折叠性”的筛选，可能适用于任何小的结合蛋白。
