KMID : 0545120200300010109
|
|
Journal of Microbiology and Biotechnology 2020 Volume.30 No. 1 p.109 ~ p.117
|
|
Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli
|
|
Kim A-Hyeon
Lee Soo-Hyun Jeon Su-Won Kim Goon-Tae Lee Eun-Jig Kim Da-Ham Kim Young-Gyu Park Tae-Sik
|
|
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.
|
|
KEYWORD
|
|
Cre recombinase, inclusion body, solubility, polyglutamate, trans-activator of transcription
|
|
FullTexts / Linksout information
|
|
|
|
Listed journal information
|
|
|