Cited 2 times in
Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli
DC Field | Value | Language |
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dc.contributor.author | 김다함 | - |
dc.contributor.author | 이은직 | - |
dc.date.accessioned | 2022-09-06T06:43:29Z | - |
dc.date.available | 2022-09-06T06:43:29Z | - |
dc.date.issued | 2020-01 | - |
dc.identifier.issn | 1017-7825 | - |
dc.identifier.uri | https://ir.ymlib.yonsei.ac.kr/handle/22282913/190293 | - |
dc.description.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 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.statementOfResponsibility | open | - |
dc.language | English | - |
dc.publisher | Korean Society for Microbiology and Biotechnology | - |
dc.relation.isPartOf | JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY | - |
dc.rights | CC BY-NC-ND 2.0 KR | - |
dc.title | Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli | - |
dc.type | Article | - |
dc.contributor.college | College of Medicine (의과대학) | - |
dc.contributor.department | Dept. of Internal Medicine (내과학교실) | - |
dc.contributor.googleauthor | A-Hyeon Kim | - |
dc.contributor.googleauthor | Soohyun Lee | - |
dc.contributor.googleauthor | Suwon Jeon | - |
dc.contributor.googleauthor | Goon-Tae Kim | - |
dc.contributor.googleauthor | Eun Jig Lee | - |
dc.contributor.googleauthor | Daham Kim | - |
dc.contributor.googleauthor | Younggyu Kim | - |
dc.contributor.googleauthor | Tae-Sik Park | - |
dc.identifier.doi | 10.4014/jmb.1909.09028 | - |
dc.contributor.localId | A00363 | - |
dc.contributor.localId | A03050 | - |
dc.relation.journalcode | J01594 | - |
dc.identifier.eissn | 1738-8872 | - |
dc.subject.keyword | Cre recombinase | - |
dc.subject.keyword | inclusion body | - |
dc.subject.keyword | solubility | - |
dc.subject.keyword | polyglutamate | - |
dc.subject.keyword | trans-activator of transcription | - |
dc.contributor.alternativeName | Kim, Daham | - |
dc.contributor.affiliatedAuthor | 김다함 | - |
dc.contributor.affiliatedAuthor | 이은직 | - |
dc.citation.volume | 30 | - |
dc.citation.number | 1 | - |
dc.citation.startPage | 109 | - |
dc.citation.endPage | 117 | - |
dc.identifier.bibliographicCitation | JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, Vol.30(1) : 109-117, 2020-01 | - |
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