Journal of Biotechnology 96 (2002) 13 – 21 Review Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology Christian Schlieker, Bernd Bukau, Axel Mogk * Institut fu ¨r Biochemie und Molekularbiologie, Hermann -Herder -Str. 7, 79104 Freiburg, Germany Received 11 July 2001; received in revised form 5 February 2002; accepted 11 February 2002 Abstract The amount of a native protein reflects an equilibrium of protein synthesis, de novo folding and protein stability. Stress situations, like heat shock, or overproduction of a protein can cause an imbalance in this equilibrium, resulting in protein aggregation. Molecular chaperones control protein folding processes and protect misfolded proteins from aggregation in all cells. Since protein aggregation is frequently observed upon synthesis of heterologous proteins in E. coli, molecular chaperones have been applied in biotechnology by their co-overproduction with the desired protein. While increasing protein solubility in some cases, this approach has not been generally successful. Recent findings demonstrate, that protein aggregation, even in case of inclusion bodies, must not be a dead end in the life cycle of a protein. Such resolubilization of aggregated proteins is mediated by a bi-chaperone system consisting of ClpB and DnaK, the prokaryotic representatives of the Hsp100 and Hsp70 families. The disaggregation capacity of this bi-chaperone system has now been demonstrated in vitro and in vivo for a wide variety of aggregated proteins and offers a new perspective to increase the solubility of proteins of interest. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Molecular chaperones; Protein aggregation; Inclusion bodies; DnaK; ClpB; Proteases www.elsevier.com/locate/jbiotec 1. Introduction Misfolding and aggregation of proteins is one of the major threats for all living organisms. Aggregation can occur during de novo folding or as a consequence of unfolding of native proteins, most commonly induced by stress. E. coli cells have evolved an elaborate protection system, con- sisting of a set of molecular chaperones, which prevent aggregation and assist refolding of mis- folded proteins. Chaperones bind to nonnative conformations of proteins and assist them to reach the native tertiary structure. Most chaper- ones bind to exposed hydrophobic surfaces of nonnative species and thereby stabilize their sub- strate against aggregation. The release of the sub- strate is often coupled to an ATP-directed conformational change in the chaperone. Follow- ing release, the substrate can proceed to fold correctly, or rebind to the chaperone again until a * Corresponding author. Fax: +49-761-2035257. E-mail address: axel.mogk@biochemie.uni-freiburg.de (A. Mogk). 0168-1656/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0168-1656(02)00033-0