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 TATCre 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 (TATCre) 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 TATCre 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.
Bibliographical noteFunding Information:
This research was supported by the Gachon University research fund of 2019 (GCU-2019-0315) and the Bio & Medical Technology Development Program through the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (NRF-2014M3A9B6069338, 2018M3A9F3020970) to T.S.P.
© 2020 by The Korean Society for Microbiology and Biotechnology.
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology