In vivo biotinylated proteins as targets for phage-display selection experiments

Scholle, Michael D.; Collart, F.; Kay, Brian K.

doi:10.1016/j.pep.2004.05.012

Cited by 41 publications

(51 citation statements)

References 49 publications

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“…Avitag is a 17-amino-acid peptide (MSGLNDIFEAQKIEWHE) that can be biotinylated by the BirA biotin ligase, and therefore can be used for immunoprecipitation of tagged proteins using streptavidin (Schatz 1993;Scholle et al 2004). This plasmid contained a neomycin selection cassette flanked by LoxP sites for positive selection and a cDNA cassette coding for diphtheria toxin chain A for negative selection.…”

Section: Prox1mentioning

confidence: 99%

The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells

Srinivasan¹,

Geng²,

Yang³

et al. 2010

Genes Dev.

250

263

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The homeobox gene Prox1 is crucial for mammalian lymphatic vascular development. In the absence of Prox1, lymphatic endothelial cells (LECs) are not specified. The maintenance of LEC identity also requires the constant expression of Prox1. However, the mechanisms controlling the expression of this gene in LECs remain poorly understood. The SRY-related gene Sox18 is required to induce Prox1 expression in venous LEC progenitors. Although Sox18 is also expressed in embryonic arteries, these vessels do not express Prox1, nor do they give rise to LECs. This finding suggests that some venous endothelial cell-specific factor is required for the activation of Prox1. Here we demonstrate that the nuclear hormone receptor Coup-TFII is necessary for the activation of Prox1 in embryonic veins by directly binding a conserved DNA domain in the regulatory region of Prox1. In addition, we show that the direct interaction between nuclear hormone receptors and Prox1 is also necessary for the maintenance of Prox1 expression during early stages of LEC specification and differentiation.[Keywords: Lymphatics; Prox1; Coup-TFII; mouse; endothelial cell] Supplemental material is available at http://www.genesdev.org.

show abstract

Section: Prox1mentioning

confidence: 99%

The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells

Srinivasan¹,

Geng²,

Yang³

et al. 2010

Genes Dev.

250

263

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“…Biotinylated proteins were cloned and expressed according to Scholle et al . [19]. Recombinant proteins were purified by immobilized metal affinity chromatography using Ni-NTA agarose.…”

Section: Methodsmentioning

confidence: 99%

“…After two additional rounds of selection, the recovered phage were serially diluted and plated to obtain single plaques. Phage enzyme linked immunosorbent assays (ELISA) were performed using a previously described protocol [19]. The DNA inserts of phage yielding positive ELISA signals were amplified by the polymerase chain reaction (PCR) using pIII (5'-TTTTTTTAGGAGATTTTCAACGTG-3') and -96 (5'-CCCTCATAGTTAGCGTAACG-3') oligonucleotides.…”

Section: Methodsmentioning

confidence: 99%

Peptide ligands specific to the oxidized form of Escherichia coli thioredoxin

Scholle

Banach

Hamdan

et al. 2008

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx’s role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5Δ22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5Δ22 with Trx, under oxidizing conditions, with an IC50 of ~10 µM.

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“…The PCRs was run using either of the plasmids pMCSG15 or pMCSG16 as template. 20 The PCR products were double-digested with NotI and BglII and gel purified on a 1% agarose gel followed by electroelution in Spectra/Por 12-14,000 kDa dialysis tube (Spectrum laboratories, Cat. No.…”

Section: Construction Of Tagsmentioning

confidence: 99%

“…One such protease is the tobacco etch virus (TEV) protease. 19,20 Because it can be difficult to predict how tags behave in a given setting, the tag combinations need to be tested for functionality and possible proteolysis during expression in the relevant host strain. 21 It is well known that E. coli contains several proteases that can degrade recombinant protein expressed in the bacteria.…”

Section: Introductionmentioning

confidence: 99%

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

Lykkemark

Mandrup

Friis

et al. 2014

mAbs

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In vivo biotinylated proteins as targets for phage-display selection experiments

Cited by 41 publications

References 49 publications

The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells

The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells

Peptide ligands specific to the oxidized form of Escherichia coli thioredoxin

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

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