In Vitro Protein Refolding

  • Daniel I. C. Wang
  • Jeffrey L. Cleland


The production of heterologous proteinsexpressed in prokaryotic organisms such as Escherichia coli often encounter the formation of inclusion bodies. Purification and reactivation of these inclusion bodies require solubilization and renaturation (Marston, 1986). During the refolding of protein from the denatured state, aggregation of partially refolded proteins occurs resulting in a decreased recovery of the native proteins. This aggregation phenomenon has been found for many proteins and by many investigators (Anfinsen and Haber, 1961; Epstein and Goldberg, 1963). The aggregation of protein during refolding is strongly dependent on the final protein and denaturant concentration (ZETTLMEISSEL et al., 1979). Due to this behavior, the in vitro refolding of proteins must be performed at low protein concentrations. The objective of this research is to examine the kinetics of protein aggregation and to devise methods to reduce the rate of protein aggregation. A model system, bovine carbonic anhydrase B (CAB), was selected for the research. This enzyme is known to aggregate during refolding (Ikai et al., 1978) and its refolding pathway has been well characterized (Stein and Henkens, 1978; Doligkh et al., 1984: Semisotnov et al. 1987).


Inclusion Body Polyethylene Glycol Guanidine Hydrochloride Denature State Protein Refold 
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Copyright information

© Springer-Verlag Tokyo 1992

Authors and Affiliations

  • Daniel I. C. Wang
    • 1
  • Jeffrey L. Cleland
    • 1
  1. 1.Department of Chemical Engineering and the Biotechnology Process Engineering CenterMassachusetts Institute of TechnologyCambridgeUSA

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