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: 2.09). 12/1998; 290:1-17. DOI: 10.1016/S0076-6879(98)90003-9
Source: PubMed


The chapter describes several common procedures for use of cell-free expression systems, including strategies to obtain homogeneous populations of nascent polypeptides, separation of ribosomal complexes, induced release of nascent polypeptides from ribosomes, and immunoadsorption of synthesized polypeptides. Factors as compactness, structural stability, cooperativity of folding, and some local structural properties of polypeptides can be studied by applying such techniques as size-exclusion chromatography under native and denaturing conditions, urea-gradient electrophoretic analysis, and limited proteolysis. The chapter presents an approach to study the kinetic mechanism of biosynthetic protein folding and assembly. The chapter also discusses approaches to evaluate in a complex mixture the equilibrium binding affinity of specific proteins. The method that is related to the enzyme-linked immunosorbent assay (ELISA) competitive binding technique, allows analysis of protein-protein interactions and, more generally, protein-ligand interactions in complex mixtures even when the interacting components are present at low levels, as is the case in cell-free expression systems. The physical and chemical principles that allow detailed descriptions of molecular interactions in comparatively simple reactions also control reactions under the complex conditions of cell-free protein synthesis.

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    • "CFPE allows studies on the complex process of genetic message transfer from DNA to protein in which a number of biomolecules and their conformational rearrangements are involved [13], [47], [48]. The addition of macromolecular crowding agents allows mimicry of the excluded volume effect of biological macromolecules in cells. "
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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.
    Full-text · Article · Dec 2011 · PLoS ONE
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    ABSTRACT: Diss. -- Helsingin yliopisto.
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    ABSTRACT: Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis -acting translational control.
    Preview · Article · Jan 1999 · Molecular and Cellular Biology
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