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: 6.72). 02/2006; DOI: 10.1039/b511736d
Source: PubMed

ABSTRACT 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: Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007. Vita. Includes bibliographical references. 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. (cont.) Paxillin is a 61-kDa protein known to orchestrate the interaction of signaling proteins involved in cell migration by acting as a molecular adaptor, with the creation of specific binding sites dependent on paxillin phosphorylation. Therefore, the semisynthetic probe comprises the entire paxillin macromolecule, including all other binding and localization domains, which are essential for creating a native-like system to probe the effect of phosphorylation. The comprehensive biochemical characterization of the paxillin probe and quantification of uncaging following irradiation with long-wavelength UV light are also described. Additionally, the strategy developed for the paxillin semisynthesis was applied to incorporate a different unnatural amino acid, the fluorescent chemosensing residue Sox, into a protein-domain sensor for ERK2 kinase activity. The protein domain sensor demonstrated significantly improved sensitivity for ERK2 phosphorylation over the corresponding peptide probe. by Elizabeth Maura Vogel. Ph.D.
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    ABSTRACT: Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed. A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation. An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.
    BMC Biotechnology 11/2008; 8(1):76. DOI:10.1186/1472-6750-8-76 · 2.59 Impact Factor
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    ABSTRACT: Protein modifications are often required to study structure and function relationships. Instead of the random labeling of lysine residues, methods have been developed to (sequence) specific label proteins. Next to chemical modifications, tools to integrate new chemical groups for bioorthogonal reactions have been applied. Alternatively, proteins can also be selectively modified by enzymes. Herein we review the methods available for site-specific modification of proteins and their applications for therapeutic antibodies.
    02/2014; 9(14):e201402001. DOI:10.5936/csbj.201402001