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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

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dc.contributor.author김다함-
dc.contributor.author이은직-
dc.date.accessioned2022-09-06T06:43:29Z-
dc.date.available2022-09-06T06:43:29Z-
dc.date.issued2020-01-
dc.identifier.issn1017-7825-
dc.identifier.urihttps://ir.ymlib.yonsei.ac.kr/handle/22282913/190293-
dc.description.abstractCre 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 TAT-Cre 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 (TAT-Cre) 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 TAT-Cre 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.-
dc.description.statementOfResponsibilityopen-
dc.languageEnglish-
dc.publisherKorean Society for Microbiology and Biotechnology-
dc.relation.isPartOfJOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY-
dc.rightsCC BY-NC-ND 2.0 KR-
dc.titleAddition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli-
dc.typeArticle-
dc.contributor.collegeCollege of Medicine (의과대학)-
dc.contributor.departmentDept. of Internal Medicine (내과학교실)-
dc.contributor.googleauthorA-Hyeon Kim-
dc.contributor.googleauthorSoohyun Lee-
dc.contributor.googleauthorSuwon Jeon-
dc.contributor.googleauthorGoon-Tae Kim-
dc.contributor.googleauthorEun Jig Lee-
dc.contributor.googleauthorDaham Kim-
dc.contributor.googleauthorYounggyu Kim-
dc.contributor.googleauthorTae-Sik Park-
dc.identifier.doi10.4014/jmb.1909.09028-
dc.contributor.localIdA00363-
dc.contributor.localIdA03050-
dc.relation.journalcodeJ01594-
dc.identifier.eissn1738-8872-
dc.subject.keywordCre recombinase-
dc.subject.keywordinclusion body-
dc.subject.keywordsolubility-
dc.subject.keywordpolyglutamate-
dc.subject.keywordtrans-activator of transcription-
dc.contributor.alternativeNameKim, Daham-
dc.contributor.affiliatedAuthor김다함-
dc.contributor.affiliatedAuthor이은직-
dc.citation.volume30-
dc.citation.number1-
dc.citation.startPage109-
dc.citation.endPage117-
dc.identifier.bibliographicCitationJOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, Vol.30(1) : 109-117, 2020-01-
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers

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