A general method for the covalent labeling of fusion proteins with small molecules in vivo (original) (raw)
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Labeling of fusion proteins with synthetic fluorophores in live cells
Proceedings of the National Academy of Sciences, 2004
A general approach for the sequential labeling of fusion proteins of O 6 -alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.
Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins
International Journal of Biological Macromolecules, 2006
Modern drug discovery strongly depends on the availability of target proteins in sufficient amounts and with desired properties. For some applications, proteins have to be produced with specific modifications such as tags for protein purification, fluorescent or radiometric labels for detection, glycosylation and phosphorylation for biological activity, and many more. It is well known that covalent modifications can have adverse effects on the biological activity of some target proteins. It is therefore one of the major challenges in protein chemistry to generate covalent modifications without affecting the biological activity of the target protein. Current procedures for modification mostly rely on non-specific labelling of lysine or cysteine residues on the protein of interest, but alternative approaches dedicated to site-specific protein modification are being developed and might replace most of the commonly used methodologies. In this study, we investigated two novel methods where target proteins can be expressed in E. coli with a fusion partner that allows protein modification in a covalent and highly selective manner. Firstly, we explored a method based on the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (hAGT) as a fusion tag for site-directed attachment of small molecules. The AGT-tag (SNAP-tag TM ) can accept almost any chemical moiety when it is attached to the guanine base through a benzyl group. In our experiments we were able to label a target protein fused to the AGT-tag with various fluorophores coupled to O 6 -benzylguanine. Secondly, we tested in vivo and in vitro site-directed biotinylation with two different tags, consisting of either 15 (AviTag TM ) or 72 amino acids (BioEase TM tag), which serve as a substrate for bacterial biotin ligase birA. When birA protein was co-expressed in E. coli biotin was incorporated almost completely into a model protein which carried these recognition tags at its C-terminus. The same findings were also obtained with in vitro biotinylation assays using pure birA independently over-expressed in E. coli and added to the biotinylation reaction in the test tube. For both biotinylation methods, peptide mapping and LC-MS proved the highly site-specific modification of the corresponding tags. Our results indicate that these novel site-specific labelling reactions work in a highly efficient manner, allow almost quantitative labelling of the target proteins, have no deleterious effect on the biological activity and are easy to perform in standard laboratories.
An Engineered Protein Tag for Multiprotein Labeling in Living Cells
Chemistry & Biology, 2008
The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O 6 -benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIPtag, which reacts specifically with O 2 -benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulsechase experiments to visualize different generations of two different proteins in one sample.
Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent
The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)-nitrilotriacetic acid (Ni 2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni 2+-NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.
Biochemistry, 1998
The Ni(II) complex of the tripeptide NH 2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc) 2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGHecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 Å of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.
The Length of Polypeptide Linker Affects the Stability of Green Fluorescent Protein Fusion Proteins
Analytical Biochemistry, 1999
The green fluorescent protein isolated from Aequorea victoria (GFP) 2 has proven to be a simple and versatile tool for the visualization of events inside the living cell (1). The vast majority of applications for GFP require the expression of fusion proteins. For these purposes GFP is appended, usually via a polypeptide linker, to the N-terminus, or the C-terminus of the protein of interest. Generally such fusions do not compromise the fluorescent properties of the GFP moiety. However, the results of our own experiments, and those of others (for example, see 2), suggest that a significant number of GFP fusion proteins are unstable in the cell, resulting in a background of GFP polypeptide no longer associated with the protein of interest, or of fusion proteins that do not fluoresce.
Protein Engineering Design and Selection, 2004
Fluorescein and its analogs are among the best¯uorophores to label proteins and the labeling generally involves chemical modi®cation of a translated protein. Using this methodology, labeling at a speci®c position remains dif®cult. It is known that the guinea pig liver transglutaminase (TGase)-catalyzed enzymatic modi®cation method can allow terminal-speci®c¯uorophore labeling of a protein by monodansylcadaverine. However, native activity of thē uorescent protein has not been investigated so far, nor has direct comparison between the chemical modi®cation and the TGase-catalyzed modi®cation been attempted. Therefore, we compared the possibility of¯uorescein labeling via chemical labeling and via TGase-catalyzed modi®cation. The latter method was found to be very practical and overcame some of the problems associated with the speci®city of the former;¯uorescein was covalently attached only to the Nor C-terminal site of glutathione S-transferase when the reaction was catalyzed by TGase and the resulting labeled protein completely retained its native activity. The TGase-mediated labeling occurred not only at room temperature but also at 4°C to the same extent, which is more desirable for preventing the inactivation of proteins.