Otten Christian, De Benedetti Stefania, Gaballah Ahmed, Bühl Henrike, Klöckner Anna, Brauner Jarryd, Sahl Hans-Georg, Henrichfreise Beate
Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.
PLoS One. 2015 Apr 7;10(4):e0122110. doi: 10.1371/journal.pone.0122110. eCollection 2015.
Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."
在大肠杆菌中外源蛋白的异源过表达常常导致错误折叠的无活性蛋白形成不溶性聚集体，即所谓的包涵体。为了解决这个问题，使用伴侣蛋白或体外重折叠程序是首选方法。由于需要额外的纯化步骤以去除伴侣蛋白或重折叠过程本身，这些方法既耗时又成本高昂。我们描述了一种易于使用的实验室规模方法来避免包涵体的形成。该方法系统地结合了通常用于生物制药制剂中生物制品体外稳定化的共溶剂的使用以及周质表达，并且可以在任何生命科学实验室中使用标准设备在一周内完成。为证明我们方法的独特效力，我们首次过量生产并纯化了一种活性衣原体青霉素结合蛋白，证明了其作为青霉素敏感DD-羧肽酶的功能，并在理解“衣原体异常”方面取得了重大进展。
