2020 ; Vol.30-1: 109~117
|Author||A-Hyeon Kim, Soohyun Lee, Suwon Jeon, Goon-Tae Kim, Eun Jig Lee, Daham Kim, Younggyu Kim, Tae-Sik Park|
|Place of duty||Department of Life Sciences, Gachon University, Sungnam 13120, Republic of Korea|
|Title||Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli|
J. Microbiol. Biotechnol.2020 ;
|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 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.|
|Key_word||Cre recombinase, inclusion body, solubility, polyglutamate,, trans-activator of transcription|
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