Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli
- Authors
- Kim, A-Hyeon; Lee, Soohyun; Jeon, Suwon; Kim, Goon-Tae; Lee, Eun Jig; Kim, Daham; Kim, Younggyu; Park, Tae-Sik
- Issue Date
- Jan-2020
- Publisher
- KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
- Keywords
- Cre recombinase; inclusion body; solubility; polyglutamate; trans-activator of transcription
- Citation
- JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, v.30, no.1, pp.109 - 117
- Journal Title
- JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
- Volume
- 30
- Number
- 1
- Start Page
- 109
- End Page
- 117
- URI
- https://scholarworks.bwise.kr/gachon/handle/2020.sw.gachon/17676
- DOI
- 10.4014/jmb.1909.09028
- ISSN
- 1017-7825
- 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.
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