Mukhopadhyay A
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Adv Biochem Eng Biotechnol. 1997;56:61-109. doi: 10.1007/BFb0103030.
Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved.
尽管重组DNA技术使生产有价值的治疗性蛋白质成为可能，但它在宿主细胞中以包涵体形式积累，给功能活性蛋白质的回收带来了严重问题。在过去的二十年里，已经开发出了替代技术来从包涵体中纯化生物活性蛋白质。其中大多数仍只是发明，只有极少数被商业利用。本综述总结了从包涵体中分离、重折叠和纯化蛋白质的进展，这些蛋白质可用于疫苗和非疫苗应用。第二部分讨论了包涵体，它们是如何形成的，以及它们的物理化学性质。第三和第四部分分别讨论了大肠杆菌中的体内蛋白质折叠和体外蛋白质折叠动力学。下一部分涵盖了从包涵体中回收生物活性蛋白质：包括从宿主细胞碎片中分离包涵体、在变性状态下纯化、交替重折叠技术以及活性分子的最终纯化。由于纯度和安全性是治疗级蛋白质的两个重要问题，接下来的三个部分专门讨论生物分子的免疫学和生物学特性、通常遇到的杂质的性质和类型以及它们的检测。最后，讨论了两个案例研究，以展示所涉及的工艺步骤顺序。
