Article

Labeling of fusion proteins of O-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

Institute of Molecular and Biological Chemistry, Ecole Polytechnique Fédérale de Lausanne, Lausanne CH-1015, Switzerland.
Methods (Impact Factor: 3.65). 05/2004; 32(4):437-44. DOI: 10.1016/j.ymeth.2003.10.007
Source: PubMed

ABSTRACT

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.

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    • "The SNAP-tag technology is based on an engineered version of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase [19] which transfers alkyl adducts from the O6-position of guanine covalently to its reactive cysteine independent of the nature of the alkyl group. The method has already been widely used for various in vivo and in vitro applications [20-22]. Particularly, labeling of SNAP tag fused septins with fluorescent Benzylguanine derivatives in living yeast cells already demonstrated that the tag and its label does not interfere with the functions of the septin subunit and its incorporation into rods and filaments [20]. "
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    ABSTRACT: The detailed understanding of the functions and mechanisms of the actin and microtubuli cytoskeleton depended, besides innovative methods in live cell imaging, on the purification and labeling of its constituents. This allowed researchers to quantitatively measure filament stability, the rates of filament turnover as well as the determination of the influence of cofactors on filament formation and structure. Septins form the least understood class of cytoskeletal structures in nearly all eukaryotic cells so far examined. In yeast, they comprise a family of proteins (Cdc3, Cdc10, Cdc11, Cdc12, Shs1) that form a co-polymeric, ring-like structure beneath the membrane. This ring serves as a template for the formation of a new bud neck and as a landing pat for proteins involved in polar growth and cytokinesis. Further progress in investigating the mechanisms of septin-structure formation and regulation is hampered by the lack of protocols to modify homogenous samples of purified septins with useful probes for in vitro biochemical studies. We present a protocol for the purification and labeling of yeast septin rods. The four individual septin subunits were co-expressed in E.coli. One subunit of the septin polymer was expressed as SNAP tag fusion protein allowing for rapid and stoichiometric labeling with derivatized Benzylguanine (BG). To demonstrate the applicability of our approach, we introduced two different SNAP tag substrates: septin rods labeled with fluorescent BG compounds enabled us to monitor the formation of filaments by fluorescence microscopy whereas BG-biotin was used to couple septin rods to a sensor chip for quantitative surface plasmon resonance binding experiments. In a first application, we determined the affinity and the binding kinetics of the yeast protein Bni5 to the individually coupled septin rods. In a further application we could demonstrate that a once formed septin rod hardly exchange its subunits. The herein introduced protocol of purifying SNAP tag modified septins from E. coli allowed us to derivatize the obtained septin rods with probes for the further in vitro characterization of this class of cytoskeletal elements. The availability of a very diverse set of SNAP tag substrates should open the way to investigate different aspects of septin biochemistry in mechanistic detail.
    Full-text · Article · Jul 2013 · BMC Biotechnology
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    • "The aim of this study was to develop a GHS-R1a binding assay which can be a reliable HTScompatible alternative to radioactivity. This assay was based on the Tag-lite technology which combines homogeneous timeresolved fluorescence (HTRF) detection with a covalent labeling technology called SNAP-tag [26] [27] [28]. The GHS-R1a fused at its N terminus with the SNAP-tag enzyme (SNAP-GHS-R1a) was expressed at the surface of living cells. "
    Dataset: leyris

    Full-text · Dataset · Nov 2012
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    • "The aim of this study was to develop a GHS-R1a binding assay which can be a reliable HTScompatible alternative to radioactivity. This assay was based on the Tag-lite technology which combines homogeneous timeresolved fluorescence (HTRF) detection with a covalent labeling technology called SNAP-tag [26] [27] [28]. The GHS-R1a fused at its N terminus with the SNAP-tag enzyme (SNAP-GHS-R1a) was expressed at the surface of living cells. "
    Dataset: leyris

    Full-text · Dataset · Nov 2012
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