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      A simple method for non-denaturing purification of biotin-tagged proteins through competitive elution with free biotin

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          Abstract

          The use of avidin or streptavidin in the purification of biotinylated proteins has been highly restricted due to the harsh and denaturing elution conditions. Here, we use biotinylated bovine serum albumin as a working model to demonstrate a simple and rapid method for biotin-tagged protein purification under non-denaturing conditions. The biotinylated bovine serum albumin is specifically bound to the anti-biotin antibody agarose beads and competitively eluted with free biotin under near-neutral conditions. The optimized elution conditions include using 4 mg/ml biotin (pH 8.5) as the elution buffer and allowing the buffer to incubate with agarose beads for 30 min prior to elution. The elution recovery rate is over 85% without apparent protein denaturation. The method is applicable for both immunoprecipitation and column chromatography.

          METHOD SUMMARY

          We describe a method for the purification of biotin-tagged proteins under non-denaturing conditions with no change in protein natural structure or function. Anti-biotin antibody agarose is employed to specifically bind the biotinylated proteins, followed by competitive elution with free biotin at near-neutral conditions. This approach is applicable for both immunoprecipitation and column chromatography.

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          Most cited references 14

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

          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 avidin/streptavidin 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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            Site-specific biotinylation of purified proteins using BirA.

            The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.
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              • Record: found
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              Antibodies to biotin enable large-scale detection of biotinylation sites on proteins

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                Author and article information

                Journal
                BTN
                BioTechniques
                Future Science Ltd (London, UK )
                0736-6205
                1940-9818
                11 December 2019
                November 2019
                : 68
                : 1
                : 41-44
                Affiliations
                1Guangxi Zhuang Autonomous Region Center for Analysis and Test Research, Nanning, Guangxi 530022, China
                2ImmuneChem Pharmaceuticals Inc., Burnaby, BC, V5J 3J1, Canada
                3Faculty of Animal Science and Technology, Gaungxi University, Nanning, Guangxi 530015, China
                Author notes
                [* ]Author for correspondence: hxiao@ 123456immunechem.com
                Article
                10.2144/btn-2019-0088
                © 2019 Hao Xiao

                This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

                Page count
                Pages: 4
                Product
                Self URI (journal page): https://www.biotechniques.com/
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