View Item 
      •   Home
      • 1. Schools
      • College of Science
      • School of Biological Sciences (SBS)
      • SBS Journal Articles
      • View Item
      •   Home
      • 1. Schools
      • College of Science
      • School of Biological Sciences (SBS)
      • SBS Journal Articles
      • View Item
      JavaScript is disabled for your browser. Some features of this site may not work without it.
      Subject Lookup

      Browse

      All of DR-NTUCommunities & CollectionsTitlesAuthorsBy DateSubjectsThis CollectionTitlesAuthorsBy DateSubjects

      My Account

      Login

      Statistics

      Most Popular ItemsStatistics by CountryMost Popular Authors

      About DR-NTU

      Butelase-mediated cyclization and ligation of peptides and proteins

      Thumbnail
      Butelase-mediated cyclization and ligation of peptides and proteins.pdf (1.566Mb)
      Author
      Nguyen, Giang Kien Truc
      Qiu, Yibo
      Cao, Yuan
      Hemu, Xinya
      Liu, Chuan-Fa
      Tam, James Pingkwan
      Date of Issue
      2016
      School
      School of Biological Sciences
      Version
      Accepted version
      Abstract
      Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His–Val at the P1′ and P2′ positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn–His–Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ~3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.
      Subject
      Butelase
      Cyclization
      Type
      Journal Article
      Series/Journal Title
      Nature Protocols
      Rights
      © 2016 Nature Publishing Group. This is the author created version of a work that has been peer reviewed and accepted for publication by Nature Protocols, Nature Publishing Group. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1038/nprot.2016.118].
      Collections
      • SBS Journal Articles
      http://dx.doi.org/10.1038/nprot.2016.118
      Get published version (via Digital Object Identifier)

      Show full item record


      NTU Library, Nanyang Avenue, Singapore 639798 © 2011 Nanyang Technological University. All rights reserved.
      DSpace software copyright © 2002-2015  DuraSpace
      Contact Us | Send Feedback
      Share |    
      Theme by 
      Atmire NV
       

       


      NTU Library, Nanyang Avenue, Singapore 639798 © 2011 Nanyang Technological University. All rights reserved.
      DSpace software copyright © 2002-2015  DuraSpace
      Contact Us | Send Feedback
      Share |    
      Theme by 
      Atmire NV
       

       

      DCSIMG