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    TSGIT : an N- and C-terminal tandem tag system for purification of native and intein-mediated ligation-ready proteins

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    pro.3989.pdf
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    3.941Mb
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    Description:
    Accepted Article
    Embargo End Date:
    2021-11-05
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    Type
    Article
    Authors
    Raducanu, Vlad-Stefan cc
    Raducanu, Daniela-Violeta
    Ouyang, Yujing
    Tehseen, Muhammad cc
    Takahashi, Masateru
    Hamdan, Samir cc
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Bioscience
    Bioscience Program
    Laboratory of DNA Replication and Recombination
    Date
    2020-11-16
    Embargo End Date
    2021-11-05
    Submitted Date
    2020-08-18
    Permanent link to this record
    http://hdl.handle.net/10754/665871
    
    Metadata
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    Abstract
    A large variety of fusion tags have been developed to improve protein expression, solubilization and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially-available expression vectors. Moreover, most commercially-available expression vectors include either N- or C-terminal fusion tags but not both. Here, we introduce TSGIT, a fusion-tag system composed of both N- and a C-terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavage tags distributed at both termini of the protein of interest. Therefore, the N- and the C-terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression and purification procedures. Each component tag is selected to maximize its benefits towards the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full-length protein is selected over truncated forms, which has been a long-standing problem in protein purification. Moreover, due to the nature of the cleavage tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N- or C-terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA-binding protein. This article is protected by copyright. All rights reserved.
    Citation
    Raducanu, V., Raducanu, D., Ouyang, Y., Tehseen, M., Takahashi, M., & Hamdan, S. M. (2020). TSGIT  : an N- and C-terminal tandem tag system for purification of native and intein-mediated ligation-ready proteins. Protein Science. doi:10.1002/pro.3989
    Publisher
    Wiley
    Journal
    Protein Science
    DOI
    10.1002/pro.3989
    PubMed ID
    33150985
    Additional Links
    https://onlinelibrary.wiley.com/doi/10.1002/pro.3989
    ae974a485f413a2113503eed53cd6c53
    10.1002/pro.3989
    Scopus Count
    Collections
    Articles; Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program

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