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Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli

Authors
 A-Hyeon Kim  ;  Soohyun Lee  ;  Suwon Jeon  ;  Goon-Tae Kim  ;  Eun Jig Lee  ;  Daham Kim  ;  Younggyu Kim  ;  Tae-Sik Park 
Citation
 JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, Vol.30(1) : 109-117, 2020-01 
Journal Title
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
ISSN
 1017-7825 
Issue Date
2020-01
Keywords
Cre recombinase ; inclusion body ; solubility ; polyglutamate ; trans-activator of transcription
Abstract
Cre recombinase is widely used to manipulate DNA sequences for both in vitro and in vivo research. Attachment of a trans-activator of transcription (TAT) sequence to Cre allows TAT-Cre to penetrate the cell membrane, and the addition of a nuclear localization signal (NLS) helps the enzyme to translocate into the nucleus. Since the yield of recombinant TAT-Cre is limited by formation of inclusion bodies, we hypothesized that the positively charged arginine-rich TAT sequence causes the inclusion body formation, whereas its neutralization by the addition of a negatively charged sequence improves solubility of the protein. To prove this, we neutralized the positively charged TAT sequence by proximally attaching a negatively charged poly-glutamate (E12) sequence. We found that the E12 tag improved the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) compared with those of TAT-NLS-Cre (TAT-Cre) when expressed in E. coli. Furthermore, the growth of cells expressing E12-TAT-Cre was increased compared with that of the cells expressing TAT-Cre. Efficacy of the purified TAT-Cre was confirmed by a recombination test on a floxed plasmid in a cell-free system and 293 FT cells. Taken together, our results suggest that attachment of the E12 sequence to TAT-Cre improves its solubility during expression in E. coli (possibly by neutralizing the ionic-charge effects of the TAT sequence) and consequently increases the yield. This method can be applied to the production of transducible proteins for research and therapeutic purposes.
Files in This Item:
T9992020518.pdf Download
DOI
10.4014/jmb.1909.09028
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers
Yonsei Authors
Kim, Daham(김다함) ORCID logo https://orcid.org/0000-0003-1871-686X
Lee, Eun Jig(이은직) ORCID logo https://orcid.org/0000-0002-9876-8370
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/190293
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