TSGIT : an N- and C-terminal tandem tag system for purification of native and intein-mediated ligation-ready proteins
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Accepted Article
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2021-11-05
Type
ArticleAuthors
Raducanu, Vlad-Stefan
Raducanu, Daniela-Violeta
Ouyang, Yujing
Tehseen, Muhammad

Takahashi, Masateru
Hamdan, Samir

KAUST Department
Biological and Environmental Sciences and Engineering (BESE) DivisionBioscience
Bioscience Program
Laboratory of DNA Replication and Recombination
Date
2020-11-16Embargo End Date
2021-11-05Submitted Date
2020-08-18Permanent link to this record
http://hdl.handle.net/10754/665871
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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.3989Publisher
WileyJournal
Protein ScienceDOI
10.1002/pro.3989PubMed ID
33150985Additional Links
https://onlinelibrary.wiley.com/doi/10.1002/pro.3989ae974a485f413a2113503eed53cd6c53
10.1002/pro.3989
Scopus Count
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