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    Two chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions

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    11-1-s2.0-S0021967320302636-main.pdf
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    PDF
    Description:
    Accepted manuscript
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    Type
    Article
    Authors
    Raducanu, Vlad-Stefan cc
    Tehseen, Muhammad cc
    Shirbini, Afnan cc
    Raducanu, Daniela-Violeta
    Hamdan, Samir cc
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Bioscience Program
    Laboratory of DNA Replication and Recombination
    Date
    2020-03-17
    Online Publication Date
    2020-03-17
    Print Publication Date
    2020-06
    Embargo End Date
    2022-03-17
    Submitted Date
    2019-11-28
    Permanent link to this record
    http://hdl.handle.net/10754/662205
    
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    Abstract
    The strength of the biotin/avidin interaction makes it an ideal tool for the purification of biotin-labeled proteins via avidin-coupled resin with high specificity and selectivity. Nevertheless, this tight binding comes at an extra cost of performing the elution step under denaturing conditions. Weakening the biotin/avidin interaction improves the elution conditions, but only to mild or harsh denaturing buffers with the drawback of reducing the specificity and selectivity of this interaction. Here, we present two chromatographic protein purification schemes that are well-suited for application under native conditions thus preserving the strength of the biotin/avidin interaction. In the first scheme, we introduce a biotin-labeled SUMO-tag to each of human flap endonuclease 1 and Escherichia coli replication termination protein Tus, and elute both proteins by performing on-resin cleavage using SUMO protease. In the second scheme, we immobilize biotin-labeled human proliferating cell nuclear antigen (PCNA) on the avidin-coupled resin and use the resulting resin as a tag-free affinity method to purify the PCNA-binding protein human DNA Ligase 1. Furthermore, we streamlined the protein biotinylation protocol by constructing a single plasmid expression system that ensures high level of expression and solubility for each of the target protein bearing the biotin-tag and the enzyme responsible for the in vivo biotinylation reaction. Both chromatographic schemes resulted in a high yield of pure proteins in their native form.
    Citation
    Raducanu, V.-S., Tehseen, M., Shirbini, A., Raducanu, D.-V., & Hamdan, S. M. (2020). Two chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions. Journal of Chromatography A, 461051. doi:10.1016/j.chroma.2020.461051
    Sponsors
    The authors would like to thank Dr. Mohamed A. Sobhy for critical reading of the manuscript and for his valuable feedback.
    This work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award [CRG3 to S.M.H.].
    Publisher
    Elsevier BV
    Journal
    Journal of Chromatography A
    DOI
    10.1016/j.chroma.2020.461051
    Additional Links
    https://linkinghub.elsevier.com/retrieve/pii/S0021967320302636
    ae974a485f413a2113503eed53cd6c53
    10.1016/j.chroma.2020.461051
    Scopus Count
    Collections
    Articles; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

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