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 | Kim, A-Hyeon | - |
dc.contributor.author | Lee, Soohyun | - |
dc.contributor.author | Jeon, Suwon | - |
dc.contributor.author | Kim, Goon-Tae | - |
dc.contributor.author | Lee, Eun Jig | - |
dc.contributor.author | Kim, Daham | - |
dc.contributor.author | Kim, Younggyu | - |
dc.contributor.author | Park, Tae-Sik | - |
dc.date.available | 2020-03-03T06:43:52Z | - |
dc.date.created | 2020-02-24 | - |
dc.date.issued | 2020-01 | - |
dc.identifier.issn | 1017-7825 | - |
dc.identifier.uri | https://scholarworks.bwise.kr/gachon/handle/2020.sw.gachon/17676 | - |
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.language | 영어 | - |
dc.language.iso | en | - |
dc.publisher | KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY | - |
dc.relation.isPartOf | JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY | - |
dc.subject | NUCLEAR-LOCALIZATION SIGNALS | - |
dc.subject | CELLULAR UPTAKE | - |
dc.subject | EXPRESSION | - |
dc.subject | PROTEIN | - |
dc.subject | TRANSDUCTION | - |
dc.subject | PEPTIDES | - |
dc.subject | DOMAIN | - |
dc.subject | YIELD | - |
dc.subject | CELLS | - |
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.type.rims | ART | - |
dc.description.journalClass | 1 | - |
dc.identifier.wosid | 000509743200014 | - |
dc.identifier.doi | 10.4014/jmb.1909.09028 | - |
dc.identifier.bibliographicCitation | JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, v.30, no.1, pp.109 - 117 | - |
dc.identifier.kciid | ART002554205 | - |
dc.identifier.scopusid | 2-s2.0-85078567917 | - |
dc.citation.endPage | 117 | - |
dc.citation.startPage | 109 | - |
dc.citation.title | JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY | - |
dc.citation.volume | 30 | - |
dc.citation.number | 1 | - |
dc.contributor.affiliatedAuthor | Kim, A-Hyeon | - |
dc.contributor.affiliatedAuthor | Jeon, Suwon | - |
dc.contributor.affiliatedAuthor | Kim, Goon-Tae | - |
dc.contributor.affiliatedAuthor | Park, Tae-Sik | - |
dc.type.docType | Article | - |
dc.subject.keywordAuthor | Cre recombinase | - |
dc.subject.keywordAuthor | inclusion body | - |
dc.subject.keywordAuthor | solubility | - |
dc.subject.keywordAuthor | polyglutamate | - |
dc.subject.keywordAuthor | trans-activator of transcription | - |
dc.subject.keywordPlus | NUCLEAR-LOCALIZATION SIGNALS | - |
dc.subject.keywordPlus | CELLULAR UPTAKE | - |
dc.subject.keywordPlus | EXPRESSION | - |
dc.subject.keywordPlus | PROTEIN | - |
dc.subject.keywordPlus | TRANSDUCTION | - |
dc.subject.keywordPlus | PEPTIDES | - |
dc.subject.keywordPlus | DOMAIN | - |
dc.subject.keywordPlus | YIELD | - |
dc.subject.keywordPlus | CELLS | - |
dc.relation.journalResearchArea | Biotechnology & Applied Microbiology | - |
dc.relation.journalResearchArea | Microbiology | - |
dc.relation.journalWebOfScienceCategory | Biotechnology & Applied Microbiology | - |
dc.relation.journalWebOfScienceCategory | Microbiology | - |
dc.description.journalRegisteredClass | scie | - |
dc.description.journalRegisteredClass | scopus | - |
dc.description.journalRegisteredClass | kci | - |
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