Intein-mediated in vitro synthesis of lipidated Ras proteins

Chair of Chemical Biology, Technische Universität Dortmund, Dortmund, North Rhine-Westphalia, Germany
Chemical Communications (Impact Factor: 6.83). 02/2006; DOI: 10.1039/b511736d
Source: PubMed


Fully functional lipid-modified Ras proteins suitable for the study of Ras-membrane interactions and embodying exclusively native amide bonds can be synthesized in preparative amounts by means of Expressed Protein Ligation.

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    ABSTRACT: Native chemical ligation (NCL) is a chemoselective reaction that joins synthetic or recombinant peptide and protein fragments through a native peptide bond. Advances in generating the required peptide and protein components for NCL have enabled the homogeneous incorporation of unnatural amino acids, fluorescent probes, and other modifications into protein domains and full-length proteins. The resulting semisynthetic proteins, which can be made in multi-milligram quantities, may be used in biophysical and biochemical studies to address challenging biological problems not accessible with traditional methods. This chapter provides an overview of NCL, including relevant design considerations and technological advances, and a discussion of recent applications that apply protein-based probes accessed through NCL.
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    ABSTRACT: Protein phosphorylation is a central regulatory mechanism in signal transduction pathways and cellular migration. Current genetic strategies for the study of phosphorylation, including gene knockout and point mutation, are limited in providing temporal information. As a complement to these techniques, the synthesis and semisynthesis of probes that enable researchers to observe the downstream effects of kinase-mediated phosphorylation in "real time" are presented in this thesis. The release of a physiologically-relevant concentration of a phosphopeptide with temporal and spatial control is accomplished by the photolysis of a photolabile precursor, a caged phosphopeptide. The synthesis and application of NI-Fmoc-protected 1-(2-nitrophenyl) ethyl (NPE) caged phosphothreonine, serine, and tyrosine building blocks facilitate the straightforward assembly of any caged phosphopeptide through Fmoc-based solid phase peptide synthesis. Removal of the NPE caging group by irradiation with long-wavelength UV light generates a concentration burst of the corresponding phosphopeptide. In addition, the installation of a caged phosphoamino acid into a full-length, multi-domain protein, the cellular migration protein paxillin, is described. A strategy, which is applicable to any expressible protein target, is detailed for the semisynthesis of a paxillin variant with a caged phosphorylated tyrosine at residue 31 of the 557-residue protein using native chemical ligation.
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    ABSTRACT: A sticky situation: A site-specific immobilization of proteins onto a glass surface may allow the creation of protein microarrays. This approach relies on the Staudinger ligation between azide-modified proteins and a phosphane-functionalized glass surface.
    Angewandte Chemie International Edition 02/2006; 45(9):1408-12. DOI:10.1002/anie.200502057 · 11.26 Impact Factor
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