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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
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- 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제30권 제1호
- 발행연도
- 2020.1
- 수록면
- 109 - 117 (9page)
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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.
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참고문헌 (34)
참고문헌 신청
Radis-Baptista G / 2017 / Cell-penetrating peptides(CPPs) : From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis / J. Biotechnol 252:15~26
Do Kwon Y / 2005 / Cellular manipulation of human embryonic stem cells by TAT-PDX1 protein transduction / Mol. Ther 12:28~32
Yang WC / 2011 / Solubility partner IF2 Domain I enables high yield synthesis of transducible transcription factors in Escherichia coli / Protein Expres. Purif 80:145~151
Xia H / 2012 / Penetratin-functionalized PEG-PLA nanoparticles for brain drug delivery / Int. J. Pharm 436:840~850
Frankel AD / 1988 / Cellular uptake of the tat protein from human immunodeficiency virus / Cell 55:1189~1193
Do Kwon Y / 2005 / Cellular manipulation of human embryonic stem cells by TAT-PDX1 protein transduction / Mol. Ther 12:28~32
Yang WC / 2011 / Solubility partner IF2 Domain I enables high yield synthesis of transducible transcription factors in Escherichia coli / Protein Expres. Purif 80:145~151
Xia H / 2012 / Penetratin-functionalized PEG-PLA nanoparticles for brain drug delivery / Int. J. Pharm 436:840~850
Frankel AD / 1988 / Cellular uptake of the tat protein from human immunodeficiency virus / Cell 55:1189~1193
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