Construction of Biotinylated Firefly Luciferases Using Biotin Acceptor Peptides

Tatsumi, Hiroki; Fukuda, Satoshi; Kikuchi, Mamoru; Koyama, Yasuji

doi:10.1006/abio.1996.0498

Cited by 58 publications

(41 citation statements)

References 4 publications

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“…Small (Ͻ23-aa) biotinylation tags have been previously obtained through multiple rounds of screening combinatorial peptide libraries for specific biotinylation by the BirA biotin ligase (16) and have been shown to be biotinylated at rates similar to those of naturally occurring substrates (17). Such peptide tags have been subsequently used for the specific biotinylation of fusion proteins in E. coli (12)(13)(14)(15). Our work shows the utility of such a small tag for the efficient BirA-mediated biotinylation of specific fusion proteins in mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, biotinylation using these tags is not very efficient, particularly in mammalian cells (10,11). Another approach, so far only demonstrated in bacteria (12)(13)(14)(15), utilizes small (Ͻ23-aa) artificial tags that have been selected through multiple rounds of screening combinatorial peptide libraries for specific biotinylation by BirA biotin ligase (16). These tags have been shown to be biotinylated in vitro with kinetics comparable to those of natural biotin acceptor sequences (17) and may thus serve as excellent substrates for efficient biotinylation in cells by coexpressed biotin ligases.…”

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confidence: 99%

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Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

Boer

Rodriguez²,

Bonte³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

398

438

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Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin͞streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

Boer

Rodriguez²,

Bonte³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

398

438

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“…Fractions containing the factors will affect the luciferin regeneration reaction. Luciferase assays have been used to determine the amounts of ATP in the various biological samples, and biotinylated luciferase has been used for immunological detection of various compounds and microorganisms (13).…”

Section: Discussionmentioning

confidence: 99%

Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin

Gomi

Kajiyama

2001

Journal of Biological Chemistry

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The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW XL . This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of D-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)؉ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5-RACE (rapid amplification of cDNA ends) and 3-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.Firefly luciferase (Luc, EC 1.13.12.7) catalyzes the oxidative decarboxylation of luciferin (LH 2 ) in the presence of ATP, O 2 , and Mg 2ϩ , producing yellow-green light ( max ϭ 560 nm) as described in the following reaction sequence:

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“…In these variants, the Thr residue at position 217 in the amino acid sequence of wild-type LcL is replaced by Ile in LcL-217I and Leu in LcL-217L. Plasmids pHLf7-217I 8) and pHLf107, 22) encoding thermostable mutants of LlL, LlL-217I and LlL-217L, were used for site-directed mutagenesis. The Ala residue at position 217 in wild-type LlL is replaced by Ile in LlL-217I and Leu in LlL-217L, in which the EcoRI site is eliminated by a silent mutation.…”

Section: Methodsmentioning

confidence: 99%

Mutant Luciferase Enzymes from Fireflies with Increased Resistance to Benzalkonium Chloride

Hattori

Kajiyama

Maeda

et al. 2002

Bioscience, Biotechnology, and Biochemistry

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Construction of Biotinylated Firefly Luciferases Using Biotin Acceptor Peptides

Cited by 58 publications

References 4 publications

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin

Mutant Luciferase Enzymes from Fireflies with Increased Resistance to Benzalkonium Chloride

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