2] Preparation and application of chaperone-deficient Escherichia coli cell-free translation systems

Abstract

Publisher Summary The chapter discusses the role of chaperones in folding of nascent proteins, either as they are synthesized on ribosomes or immediately after they are terminated and released. Five bacterial chaperones—DnaJ, DnaK, GrpE, GroEL, and GroES—can promote folding and release of full-length rhodanese that was not properly folded and released from Escherichia coli ( E. coli ) ribosomes after its synthesis. The chapter discusses whether this is the major pathway by which nascent proteins are folded into their native conformation. E. coli salt-washed ribosomes, their 50S subunit, or even the 23S rRNA in its native state can efficiently refold denatured rhodanese independent of Adenosine-5'-triphosphate (ATP). The results relate the ability of ribosomes to carry out refolding to the peptidyltransferase center and their ability to form peptide bonds. To test the hypothesis that ribosomes themselves can promote folding of nascent proteins without the assistance of molecular chaperones, in vitro systems for protein synthesis that are deficient in molecular chaperones are developed. The aim is to develop a system that totally lacks these proteins. The description of the experimental procedures addresses the following three aspects: (1) preparation of the cell-free systems, (2) methods to quantitate the chaperones that remain in these systems, and (3) in vitro synthesis of test proteins and their analysis, including their enzymatic activity.