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Site-specific protein labeling with SNAP-tags.

ABSTRACT: Site-specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP-tag is a modified form of the DNA repair enzyme human O(6)-alkylguanine-DNA-alkyltransferase, and undergoes a self-labeling reaction to form a covalent bond with O(6)-benzylguanine (BG) derivatives. BG can be modified with a wide variety of fluorophores and other reporter compounds, generally without affecting the reaction with the SNAP-tag. In this unit, basic strategies for labeling SNAP-tag fusion proteins, both for live cell imaging and for in vitro analysis, are described. This includes a description of a releasable SNAP-tag probe that allows the user to chemically cleave the fluorophore from the labeled SNAP-tag fusion. In vitro labeling of purified SNAP-tag fusions is briefly described.

SUBMITTER: Cole NB

PROVIDER: S-EPMC3920298 | biostudies-other | 2013 Sep

REPOSITORIES: biostudies-other

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Site-specific protein labeling with SNAP-tags.

Cole Nelson B NB

Current protocols in protein science 20130924

Site-specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP-tag is a modified form of the DNA repair enzyme human O(6)-alkylguanine-DNA-alkyltransferase, and undergoes a self-labeling reaction to form a covale ...[more]

PMID: 24510614

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Project description:Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

Dataset Information

Site-specific protein labeling with SNAP-tags.

Publications

Site-specific protein labeling with SNAP-tags.

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