Protein derivitization-expressed protein ligation

Sarah F. Mitchell, Jon R. Lorsch

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Expressed protein ligation (EPL) combines two methods to ligate a synthetic peptide to a recombinant protein. Native chemical ligation (NCL) is a process in which two synthesized peptides are ligated by reaction of a C-terminal thioester on one peptide with an N-terminal cysteine residue of another protein. The chemistry of inteins, self-excising protein fragments that ligate the surrounding protein back together, creates isolatable intermediates with the two chemical groups necessary for NCL, a C-terminal thioester and an N-terminal cysteine residue. This technique allows for the incorporation of synthetic amino acids, radiolabeled amino acids, and fluorescent moieties at specific locations in a protein. It has the advantage of allowing attachment of such synthetic peptides to the termini of a recombinant protein, allowing for the synthesis of large proteins with modified amino acids. This technique utilizes the IMPACTTM-System created by New England Biolabs, who provide a variety of vectors in which the multicloning site is directly upstream of an intein sequence fused to a chitin-binding domain (CBD). The CBD binds tightly and specifically to chitin beads, allowing for an efficient one-step purification. This step can be used to obtain highly purified proteins (see Protein Affinity Purification using Intein/Chitin Binding Protein Tags). After purification of the recombinant protein, cleavage from the intein is achieved through the addition of a reactive thiol compound, usually sodium 2-mercaptoethanesulfonate (MESNA) (see also Proteolytic affinity tag cleavage). This reaction creates a protein with a C-terminal thioester that can then react with a peptide containing an N-terminal cysteine residue, ligating the two proteins via a peptide bond.

Original languageEnglish (US)
Title of host publicationLaboratory Methods in Enzymology
Subtitle of host publicationProtein Part A
PublisherAcademic Press Inc.
Pages95-108
Number of pages14
ISBN (Print)9780124200708
DOIs
StatePublished - Jan 1 2014

Publication series

NameMethods in Enzymology
Volume536
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Cell harvesting
  • Cell lysis
  • Chitin beads and linking peptide
  • Chitin-binding domain (CBD)
  • Elution and characterization of protein
  • Expressed protein ligation (EPL)
  • Peptide

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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