Protein Folding In Vivo and Renaturation of Recombinant Proteins from Inclusion Bodies Andrew D. Guise, Shauna M. West, and Julian B. Chaudhuri* Abstract Eukaryotic proteins expressed in Escheriehia coli often accumulate within the ceil as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion:bodies iS a complex processi there are no general rules for efficient renaturation. Research into understan~ng how proteins fold in Vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones, In this article we review what is understood about the mMn three classes of chaperone: the Stress 60, Stress 70, and Index Entries: Protein refolding; molecular chaperones; inclusion bodies; multisubunits; polyethylene glycol: arginine. 1. Introduction The overexpression of recombinant proteins in E. coli can result in the formation of inclusion bodies in the cell cytoplasm or periplasm (1,2). Inclusion bodies have also been observed in gram- positive and gram-negative bacteria, in Saccharo- myces cerevisiae and in insect cells (3). Formation of inclusion bodies in mammalian or plant cells appears to be a rare phenomenon. Several recom- binant proteins on the market are produced as inclu- sion bodies, these include human growth hormone, human insulin, and granulocycte-colony stimulat- ing factor (G-CSF). Inclusion bodies are insoluble aggregates of the recombinant protein, which are visible by light microscope and appear refractile to light. There is generally one inclusion body per bacterial cell and the total mass and number of inclusion bodies are proportional to the product level. The inclusion body protein is misfolded and has no biological activity: Thus there is a need to refold the inclusion body to its native structure in order to regain bio- logical activity. Inclusion bodies are formed from the accumula- tion of folding intermediates rather than from the native or unfolded polypeptide. Three reasons have been suggested to account for the formation of inclu- sion bodies when eukaryotic proteins are expressed in E. coli (4): The presence of the protein secretory sequence may obstruct folding resulting in a mis- folded molecule; the cytoplasm is a very reducing environment that may decrease the stabilizing effect of the disulfide bonds; and the inability of E. coli to glycosylate the protein may reduce the solubility of the intermediate resulting in insoluble aggregates. It is not possible to generalize or predict which pro- teins will be produced as inclusion bodies. In a study on the expression of proteins from bacteria, yeast, virus, and animal cells in E. coli, no relationship was observed between the formation of intracellular aggregates and the origin of the protein, the promoter used, and the hydrophobicity of the polypeptide (2). Fermentation temperature is a major factor dictating the formation of inclusion bodies; growth at lower temperatures may result in an active soluble protein rather than an aggregate (5). *Author to whom all correspondence and reprint requests should be addressed. School of Chemical Engineering, University of Bath BA2 7AY UK, E-mail: j.b.chaudhuri@bath.ac.uk. Molecular Biotechnology 9 Humana Press Inc. All rights of any nature whatsoever reserved. 1073-6085/1996/6:1/53-64/$8.00 !!!z !iz:! 84 ~ii iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii i il ii i i i i iiiiiii 84 ! !! !!!!!!!!!!i 84 i i!iiiiiiiiii ii iiiiii!!!!!ii!! !!!i !i!!! i i i i i i iiiiiiiiiiiiiii!!!!i!i!iii!!i! ! iii ii i i !!!!