Optimal screening of surface-displayed polypeptide libraries
Optimal screening of surface-displayed polypeptide libraries
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DOI：
10.1021/bp970144q
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发表时间：
1998-01-01
影响因子：
2.9
通讯作者：
Wittrup, KD
中科院分区：
文献类型：
--
作者：
Boder, ET;Wittrup, KD
多肽文库的细胞表面显示与流式细胞术细胞分选相结合，具有显著的增强蛋白质配体识别特性的潜力。为了使该方法的效用最大化，必须优化筛选和纯化条件，以充分利用该技术的定量特征。特别是，从过量的野生型多肽中区分改进的文库突变体依赖于有效的筛选方法。利用荧光配体筛选的多肽文库的期望细胞荧光强度的数学模型推导出改进的文库突变体的荧光识别谱。表面显示的文库在平衡和动力学筛选条件下的分布具有明显的判别最优，并由此推导出最优的平衡和动力学筛选参数。此外，流式细胞术分析细胞群的统计模型表明，低严格分选的重要性，然后通过再生和增加严格程度进行扩增。该分析进一步对细胞分选的严格程度提出了定量建议。
Cell surface display of polypeptide libraries combined with flow cytometric cell sorting presents remarkable potential for enhancement of protein-ligand recognition properties. To maximize the utility of this approach, screening and purification conditions must be optimized to take full advantage of the quantitative feature of this technique. In particular, discrimination of improved library mutants from an excess of wild-type polypeptides is dependent upon an effective screening methodology. Fluorescence discrimination profiles for improved library mutants were derived from a mathematical model of expected cell fluorescence intensities for polypeptide libraries screened with fluorescent ligand. Profiles for surface-displayed libraries under equilibrium or kinetic screening conditions demonstrate distinct discrimination optima from which optimal equilibrium and kinetic screening parameters were derived. In addition, a statistical model of flow cytometrically analyzed cell populations indicates the importance of low-stringency sorting followed by amplification through regrowth and resorting at increased stringency. This analysis further yields quantitative recommendations for cell-sorting stringency.