化学
变性(裂变材料)
荧光光谱法
苯丙氨酸羟化酶
蛋白质聚集
色谱法
差示扫描量热法
等温过程
苯丙氨酸
荧光
生物化学
核化学
氨基酸
物理
量子力学
热力学
作者
Paula Leandro,Paulo Roque Lino,Raquel Lopes,João Leandro,Mariana Amaro,Paulo Gleisson Rodrigues de Sousa,João B. Vicente,António Almeida
标识
DOI:10.1016/j.ejpb.2023.03.012
摘要
The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.
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