Expressed protein ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase: An application to a protein expressed as an inclusion body SCIE SCOPUS KCI

DC Field Value Language
dc.contributor.author Kim, Hak Jun -
dc.contributor.author Shin, Hee Jae -
dc.contributor.author Kim, Hyun Woo -
dc.contributor.author Kang, Sung-Ho -
dc.contributor.author Kim, Young Tae -
dc.date.accessioned 2020-04-20T11:40:08Z -
dc.date.available 2020-04-20T11:40:08Z -
dc.date.created 2020-01-28 -
dc.date.issued 2007-12-20 -
dc.identifier.issn 0253-2964 -
dc.identifier.uri https://sciwatch.kiost.ac.kr/handle/2020.kiost/4605 -
dc.description.abstract Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its C-terminal fragment (EPSPSC, residues Mee(237)-238(CYS)-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing similar to 1 mM of EPSPSN-thioester with similar to 2 mM of EPSPSCCYS (residues 238(CYS)-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was similar to 85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and similar to 115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins. -
dc.description.uri 1 -
dc.language English -
dc.publisher KOREAN CHEMICAL SOC -
dc.subject YEAST PHOSPHOGLYCERATE KINASE -
dc.subject SITE-DIRECTED MUTAGENESIS -
dc.subject CHEMICAL LIGATION -
dc.subject ESCHERICHIA-COLI -
dc.subject 3-PHOSPHATE SYNTHASE -
dc.subject TERMINAL DOMAIN -
dc.subject HERBICIDE GLYPHOSATE -
dc.subject UNPROTECTED PEPTIDES -
dc.subject RECOMBINANT PROTEINS -
dc.subject SEMISYNTHESIS -
dc.title Expressed protein ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase: An application to a protein expressed as an inclusion body -
dc.type Article -
dc.citation.endPage 2309 -
dc.citation.startPage 2303 -
dc.citation.title BULLETIN OF THE KOREAN CHEMICAL SOCIETY -
dc.citation.volume 28 -
dc.citation.number 12 -
dc.contributor.alternativeName 신희재 -
dc.identifier.bibliographicCitation BULLETIN OF THE KOREAN CHEMICAL SOCIETY, v.28, no.12, pp.2303 - 2309 -
dc.identifier.scopusid 2-s2.0-38049127376 -
dc.identifier.wosid 000252629100026 -
dc.type.docType Article -
dc.identifier.kciid ART001214736 -
dc.description.journalClass 1 -
dc.subject.keywordPlus YEAST PHOSPHOGLYCERATE KINASE -
dc.subject.keywordPlus SITE-DIRECTED MUTAGENESIS -
dc.subject.keywordPlus CHEMICAL LIGATION -
dc.subject.keywordPlus ESCHERICHIA-COLI -
dc.subject.keywordPlus 3-PHOSPHATE SYNTHASE -
dc.subject.keywordPlus TERMINAL DOMAIN -
dc.subject.keywordPlus HERBICIDE GLYPHOSATE -
dc.subject.keywordPlus UNPROTECTED PEPTIDES -
dc.subject.keywordPlus RECOMBINANT PROTEINS -
dc.subject.keywordPlus SEMISYNTHESIS -
dc.subject.keywordAuthor expressed protein ligation -
dc.subject.keywordAuthor intein -
dc.subject.keywordAuthor 5-Enolpyruvylshikimate-3-phosphate synthase -
dc.subject.keywordAuthor methionine aminopeptidase -
dc.relation.journalWebOfScienceCategory Chemistry, Multidisciplinary -
dc.description.journalRegisteredClass scie -
dc.description.journalRegisteredClass scopus -
dc.description.journalRegisteredClass kci -
dc.relation.journalResearchArea Chemistry -
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Marine Resources & Environment Research Division > Marine Biotechnology &Bioresource Research Department > 1. Journal Articles
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