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Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate.

ABSTRACT: Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.

SUBMITTER: Sawasaki T

PROVIDER: S-EPMC7164004 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate.

Sawasaki Tatsuya T Kamura Nami N Matsunaga Satoko S Saeki Mihoro M Tsuchimochi Masateru M Morishita Ryo R Endo Yaeta Y

FEBS letters 20071217 2

Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition ...[more]

PMID: 18082144

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Project description:Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label, or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this approach for polypeptide modification could be particularly useful for large-scale production of protein conjugates for therapeutic or industrial applications.

Dataset Information

Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate.

Publications

Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate.

Similar Datasets

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