Metabolic Biotinylation of Secreted and Cell Surface Proteins from Mammalian Cells

Parrott, M. Brandon; Barry, Michael A.

doi:10.1006/bbrc.2001.4437

Cited by 66 publications

(55 citation statements)

References 20 publications

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“…Our work shows the utility of such a small tag for the efficient BirA-mediated biotinylation of specific fusion proteins in mammalian cells. Larger (Ͼ63-aa) tags derived from biotin acceptor domains present in naturally biotinylated proteins have been previously used in biotinylating fusion proteins in mammalian cells (10,11). However, there are obvious advantages in using smaller artificial tags.…”

Section: Discussionmentioning

confidence: 99%

“…The characterization of the minimal amino acid sequence requirements of naturally biotinylated proteins has led to the development of sequence tags that can be biotinylated in bacterial, yeast, insect, and mammalian cells (7)(8)(9)(10)(11). Biotinylation can occur either by the cell's endogenous protein-biotin ligases or through the coexpression of an exogenous biotin ligase, in most cases that of the bacterial BirA enzyme.…”

mentioning

confidence: 99%

“…In addition, that these tags can be recognized by endogenous enzymes excludes applications where biotinylation of the tagged protein may need to be regulated. 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).…”

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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.

401

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%

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confidence: 99%

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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.

401

438

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“…Bait proteins were enzymatically biotinylated during synthesis by co-transfecting an expression plasmid containing a modified E. coli biotin ligase, BirA, together with the bait expression construct in a 1:9 ratio. The BirA coding sequence was fused downstream of the rat Cd4 5Ј UTR and signal peptide to direct expression to the secretory pathway essentially as described by Parrott and Barry (2001). When the culture medium was supplemented with 100 µM D-biotin, at least 95% of the target protein was biotinylated (data not shown).…”

Section: Bait Protein Expressionmentioning

confidence: 99%

Large-scale screening for novel low-affinity extracellular protein interactions

Söllner²,

et al. 2008

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Extracellular protein-protein interactions are essential for both intercellular communication and cohesion within multicellular organisms. Approximately a fifth of human genes encode membrane-tethered or secreted proteins, but they are largely absent from recent large-scale protein interaction datasets, making current interaction networks biased and incomplete. This discrepancy is due to the unsuitability of popular high-throughput methods to detect extracellular interactions because of the biochemical intractability of membrane proteins and their interactions. For example, cell surface proteins contain insoluble hydrophobic transmembrane regions, and their extracellular interactions are often highly transient, having half-lives of less than a second. To detect transient extracellular interactions on a large scale, we developed AVEXIS (avidity-based extracellular interaction screen), a high-throughput assay that overcomes these technical issues and can detect very transient interactions (halflives Յ 0.1 sec) with a low false-positive rate. We used it to systematically screen for receptor-ligand pairs within the zebrafish immunoglobulin superfamily and identified novel ligands for both well-known and orphan receptors. Genes encoding receptor-ligand pairs were often clustered phylogenetically and expressed in the same or adjacent tissues, immediately implying their involvement in similar biological processes. Using AVEXIS, we have determined the first systematic low-affinity extracellular protein interaction network, supported by independent biological data. This technique will now allow large-scale extracellular protein interaction mapping in a broad range of experimental contexts.

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“…BirA has previously been used to biotinylate AP-tagged proteins in vitro or when expressed in the cytosol (11,12). BirA also biotinylates a truncated version of a bacterial transcarboxylase, when enzyme and substrate are coexpressed in the mammalian secretory pathway (13). The mammalian enzyme with biotin ligase activity, holocarboxylase synthetase (14), does not recognize AP as a substrate (12).…”

mentioning

confidence: 99%

Targeting quantum dots to surface proteins in living cells with biotin ligase

Howarth

Takao

Hayashi

et al. 2005

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

523

463

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Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.BirA ͉ labeling ͉ streptavidin ͉ glutamate receptor ͉ single molecule

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Metabolic Biotinylation of Secreted and Cell Surface Proteins from Mammalian Cells

Cited by 66 publications

References 20 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

Large-scale screening for novel low-affinity extracellular protein interactions

Targeting quantum dots to surface proteins in living cells with biotin ligase

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