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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

Journal of Microbiology and Biotechnology 2020년 30권 1호 p.109 ~ 117
 ( Kim A-Hyeon ) - Gachon University Department of Life Science

 ( Lee Soo-Hyun ) - LumiMac Inc.
 ( Jeon Su-Won ) - Gachon University Department of Life Science
김균태 ( Kim Goon-Tae ) - Gachon University Department of Life Science
이은직 ( Lee Eun-Jig ) - Yonsei University College of Medicine Department of Internal Medicine
김다함 ( Kim Da-Ham ) - Yonsei University College of Medicine Department of Internal Medicine
 ( Kim Young-Gyu ) - LumiMac Inc.
박태식 ( Park Tae-Sik ) - Gachon University Department of Life Science

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.

키워드

Cre recombinase; inclusion body; solubility; polyglutamate; trans-activator of transcription
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