TSGIT : an N- and C-terminal tandem tag system for purification of native and intein-mediated ligation-ready proteins
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Laboratory of DNA Replication and Recombination
Embargo End Date2021-11-05
Permanent link to this recordhttp://hdl.handle.net/10754/665871
MetadataShow full item record
AbstractA 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.
CitationRaducanu, 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
- Ligation-independent cloning and self-cleaving intein as a tool for high-throughput protein purification.
- Authors: Warren TD, Coolbaugh MJ, Wood DW
- Issue date: 2013 Oct
- Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification.
- Authors: Wang Z, Li N, Wang Y, Wu Y, Mu T, Zheng Y, Huang L, Fang X
- Issue date: 2012 Mar
- Systematic analysis of the expression, solubility and purification of a passenger protein in fusion with different tags.
- Authors: Bernier SC, Cantin L, Salesse C
- Issue date: 2018 Dec
- A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production.
- Authors: Cabrita LD, Dai W, Bottomley SP
- Issue date: 2006 Mar 1
- Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins.
- Authors: Zhang A, Gonzalez SM, Cantor EJ, Chong S
- Issue date: 2001 Sep 19