Mild conditions for releasing mono and bis-biotinylated macromolecules from immobilized streptavidin

Nguyen, Giang Huong; Milea, Jaqueline S.; Rai, Anurag; Smith, Cassandra L.

doi:10.1016/j.bioeng.2005.02.003

Cited by 12 publications

(8 citation statements)

References 16 publications

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“…In particular, the addition of an incubation step at room temperature after the PCR leads to a 150-fold increase in the number of DNA copies per bead (Chivers et al , 2010). This step is likely to increase the capture of BB PCR products to the streptavidin-coated beads (Holmberg et al , 2005; Nguyen et al , 2005). Furthermore, a previous protocol employed a covalently bound primer, leading to inefficient ePCR (Paul et al , 2013).…”

Section: Resultsmentioning

confidence: 99%

In vitro affinity screening of protein and peptide binders by megavalent bead surface display

Diamante

Gatti‐Lafranconi

Schaerli

et al. 2013

Protein Engineering Design and Selection

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The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 106 of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 103 and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead Kd measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

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Section: Resultsmentioning

confidence: 99%

In vitro affinity screening of protein and peptide binders by megavalent bead surface display

Diamante

Gatti‐Lafranconi

Schaerli

et al. 2013

Protein Engineering Design and Selection

View full text Add to dashboard Cite

show abstract

“…Slightly less rAR was recovered using the “SDS/Biotin” buffer (lane 2), with subsequent control elution of beads yielding additional rAR signal. Though a previous study has reported elution of biotinylated oligonucleotides from magnetic streptavidin beads (Invitrogen M-280 Dynabeads) with TE buffer (10 mM Tris pH 8, 1 mM EDTA) and biotin alone [35], no elution was achieved from the streptavidin beads in the absence of SDS using “biotin” buffer (lane 3), while subsequent control elution of the beads released the rAR protein. The small loss in recovery observed between the “urea/SDS/biotin” and “SDS/biotin” buffers is likely due to the absence of urea, which alters the streptavidin quaternary structure and acts as a biotin analog at millimolar concentrations [36].…”

Section: Resultsmentioning

confidence: 99%

Proteomic analysis of the androgen receptor via MS‐compatible purification of biotinylated protein on streptavidin resin

et al. 2011

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The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high temperature elution in urea containing buffers, we show mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde crosslinked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.

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“…12 Similarly, Nguyen et al demonstrated the release of multibiotinylated molecules from streptavidin in the presence of biotin and high temperatures. 13 However, the release of biotinylated molecules was incomplete. 13 Trials to change the properties of (strept)avidin to enable release of biotin under mild conditions by modifying the biotin-binding pocket resulted in a biotin affinity that was too low, 14 an incomplete modification due to the chemical approach, 15 or a high dissociation rate.…”

Section: ■ Introductionmentioning

confidence: 99%

Switchavidin: Reversible Biotin–Avidin–Biotin Bridges with High Affinity and Specificity

et al. 2014

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Switchavidin is a chicken avidin mutant displaying reversible binding to biotin, an improved binding affinity toward conjugated biotin, and low nonspecific binding due to reduced surface charge. These properties make switchavidin an optimal tool in biosensor applications for the reversible immobilization of biotinylated proteins on biotinylated sensor surfaces. Furthermore, switchavidin opens novel possibilities for patterning, purification, and labeling.

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Mild conditions for releasing mono and bis-biotinylated macromolecules from immobilized streptavidin

Cited by 12 publications

References 16 publications

In vitro affinity screening of protein and peptide binders by megavalent bead surface display

In vitro affinity screening of protein and peptide binders by megavalent bead surface display

Proteomic analysis of the androgen receptor via MS‐compatible purification of biotinylated protein on streptavidin resin

Switchavidin: Reversible Biotin–Avidin–Biotin Bridges with High Affinity and Specificity

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