Williams Cameron, Dougherty Melissa L, Makaroff Katherine, Stapleton Jacob, Konkolewicz Dominik, Berberich Jason A, Page Richard C
Miami University, Oxford, OH, United States.
Miami University, Oxford, OH, United States.
Methods Enzymol. 2017;590:93-114. doi: 10.1016/bs.mie.2016.11.008. Epub 2017 Jan 10.
Protein-polymer conjugates are increasingly viewed as promising avenues to producing industrial enzymes with high activity capable of withstanding potentially harsh reaction conditions, or to designing novel therapeutics with triggered release, controlled masking, or increased resistance to proteolytic degradation. Common among these applications are the desire to improve the stability of protein-polymer conjugates to unfolding by exposure to chemicals or thermal stress. Thus, assays that allow researchers to robustly and easily characterize protein-polymer conjugates by obtaining thermodynamic parameters for folding stand to play an important role in the development of improved protein-polymer conjugates. Herein, we describe two techniques, differential scanning fluorimetry and intrinsic tryptophan fluorescence, used in our laboratories to obtain thermodynamic parameters of unfolding that allow for direct comparison of protein-polymer conjugates and the myriad effects of variations in attachment site, polymer identity, and polymer length. These two experiments, which are easily amenable to parallelization, are presented as high-throughput replacements for more traditionally employed circular dichroism experiments and as complements to functional chemical stability or functional thermal stability experiments. Each assay is presented in a parallelized format that allows for rapid scaling and high-throughput analysis of protein-polymer conjugate libraries. Descriptions of the assays include a discussion of advantages and disadvantages alongside protocol details and approaches to data analysis.
蛋白质-聚合物缀合物越来越被视为一种有前景的途径，可用于生产具有高活性、能够耐受潜在苛刻反应条件的工业酶，或用于设计具有触发释放、可控屏蔽或增强抗蛋白水解降解能力的新型治疗药物。这些应用的共同之处在于希望提高蛋白质-聚合物缀合物在暴露于化学物质或热应激时的抗解折叠稳定性。因此，能够让研究人员通过获取折叠的热力学参数来可靠且轻松地表征蛋白质-聚合物缀合物的分析方法，在改进蛋白质-聚合物缀合物的开发中有望发挥重要作用。在此，我们描述了两种技术，差示扫描荧光法和色氨酸固有荧光法，它们在我们实验室中用于获取解折叠的热力学参数，从而能够直接比较蛋白质-聚合物缀合物以及连接位点、聚合物特性和聚合物长度变化所产生的各种影响。这两个易于并行化的实验，作为对更传统使用的圆二色性实验的高通量替代方法以及对功能化学稳定性或功能热稳定性实验的补充方法被呈现。每个分析方法都以并行化形式呈现，允许对蛋白质-聚合物缀合物文库进行快速扩展和高通量分析。分析方法的描述包括对优缺点的讨论以及实验方案细节和数据分析方法。
