Singh Anupam, Upadhyay Vaibhav, Singh Akansha, Panda Amulya K
Product Development Cell, National Institute of Immunology, New Delhi, India.
Front Microbiol. 2020 May 8;11:876. doi: 10.3389/fmicb.2020.00876. eCollection 2020.
High level expression of recombinant proteins in bacteria often results in their aggregation into inclusion bodies. Formation of inclusion bodies poses a major bottleneck in high-throughput recovery of recombinant protein. These aggregates have amyloid-like nature and can retain biological activity. Here, effect of expression temperature on the quality of asparaginase II (a tetrameric protein) inclusion bodies was evaluated. Asparaginase was expressed as inclusion bodies at different temperatures. Purified inclusion bodies were checked for biological activities and analyzed for structural properties in order to establish a structure-activity relationship. Presence of activity in inclusion bodies showed the existence of properly folded asparaginase tetramers. Expression temperature affected the properties of asparaginase inclusion bodies. Inclusion bodies expressed at higher temperatures were characterized by higher biological activity and less amyloid content as evident by Thioflavin T binding and Fourier Transform Infrared (FTIR) spectroscopy. Complex kinetics of proteinase K digestion of asparaginase inclusion bodies expressed at higher temperatures indicate higher extent of conformational heterogeneity in these aggregates.
重组蛋白在细菌中的高水平表达常常导致其聚集成包涵体。包涵体的形成是重组蛋白高通量回收的一个主要瓶颈。这些聚集体具有类淀粉样性质并且能够保留生物活性。在此，评估了表达温度对天冬酰胺酶II（一种四聚体蛋白）包涵体质量的影响。天冬酰胺酶在不同温度下表达为包涵体。对纯化的包涵体进行生物活性检查并分析其结构性质，以建立结构 - 活性关系。包涵体中活性的存在表明存在正确折叠的天冬酰胺酶四聚体。表达温度影响天冬酰胺酶包涵体的性质。较高温度下表达的包涵体具有较高的生物活性和较少的淀粉样蛋白含量，这通过硫黄素T结合和傅里叶变换红外（FTIR）光谱法得以证明。较高温度下表达的天冬酰胺酶包涵体蛋白酶K消化的复杂动力学表明这些聚集体中构象异质性程度更高。
