Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering
CHAPTER 2.2: In vitro Refolding of Proteins
Published:27 Jul 2018
Special Collection: 2018 ebook collectionSeries: Chemical Biology
M. Haslbeck and J. Buchner, in Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, ed. M. J. Feige, The Royal Society of Chemistry, 2018, pp. 129-151.
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Recombinant protein expression in the cytosol of Escherichia coli represents a standard low-cost and high-yield production process in academia and industry. However, in vivo, the solubility of the recombinant proteins is often limited. Approximately half of the proteins overexpressed in E. coli are insoluble and require solubilization and subsequent refolding. This refolding step represents a significant bottleneck in process development as suitable refolding conditions have to be determined by trial and error, leading many scientists to avoid these approaches and leaving many highly interesting but aggregation-prone target proteins unexplored. This chapter describes the basic principles of in vitro protein refolding and reoxidation approaches. Additionally, standardized protocols for solubilization and refolding of insolubly expressed proteins and also strategies to screen efficiently for optimum refolding conditions are provided.