Protein folding and assembly in a cell-free expression system

Department of Biochemistry, Texas A&M University, College Station 77843-2128, USA.
Methods in Enzymology (Impact Factor: 2.19). 01/1998; 290:1-17. DOI: 10.1016/S0076-6879(98)90003-9
Source: PubMed
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    ABSTRACT: Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process. Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments. Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.
    PLoS ONE 12/2011; 6(12):e28707. DOI:10.1371/journal.pone.0028707 · 3.53 Impact Factor
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    ABSTRACT: A new method that uses a combination of bacterial GroE chaperonins and cellular osmolytes for in vitro protein folding is described. With this method, one can form stable chaperonin–protein folding intermediate complexes to prevent deleterious protein aggregation and, using these complexes, screen a large array of osmolyte solutions to rapidly identify the superior folding conditions. As a test substrate, we used GSΔ468, a truncation mutant of bacterial glutamine synthetase (GS) that cannot be refolded to significant yields in vitro with either chaperones or osmolytes alone. When our chaperonin/osmolyte method was employed to identify and optimize GSΔ468 refolding conditions, 67% of enzyme activity was recovered, comparable with refolding yields of wild type GS. This method can potentially be applied to the refolding of a broad spectrum of proteins. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 1036–1045, 2000
    Journal of Pharmaceutical Sciences 08/2000; 89(8):1036-1045. DOI:10.1002/1520-6017(200008)89:83.0.CO;2-5 · 3.01 Impact Factor