The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation,ACS Synthetic Biology

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The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-01-22 00:00:00 , DOI: 10.1021/acssynbio.7b00316
Kristen M. Wilding ₁ , Addison K. Smith ₁ , Joshua W. Wilkerson ₁ , Derek B. Bush ₁ , Thomas A. Knotts ₁ , Bradley C. Bundy ₁

Affiliation

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.

中文翻译：

特定于站点的聚乙二醇化的位置影响：具有无细胞蛋白表达和粗粒模拟的简化筛选

尽管通常使用聚乙二醇（PEG）来改善蛋白质的稳定性和治疗功效，但尚未很好地了解将PEG附着在蛋白质上的最佳位置。在这里，我们介绍了一个基于无细胞蛋白质合成的筛选平台，该平台可促进位点特异性PEG化，并有效评估PEG附着效率，热稳定性和PEG化T4溶菌酶不同变体（包括二聚PEG化变体）的活性。我们还报告了开发一种计算效率高的粗粒度模拟模型，作为缩小实验筛选候选对象的潜在工具。我们使用这种模拟方法作为一种新颖的工具来评估聚乙二醇化的位置影响。使用此屏幕，我们还评估了聚乙二醇化位点溶剂可及性，结合位点结构，聚乙二醇大小，和双重PEG化。我们的发现表明，聚乙二醇化效率，蛋白质稳定性和蛋白质活性随聚乙二醇化位点的不同而有很大差异，而常见的聚乙二醇化指南并未很好地预测这些变异。总体而言，我们的结果表明，当前的指南对预测的预测不足，突出了对实验和模拟筛选系统（如此处介绍的系统）的需求。

更新日期：2018-01-22

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