title: Protein derivitization-expressed protein ligation.
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 IMPACT(TM)-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.
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Mitchell
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: jlorsch@jhmi.edu.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
mesh_terms: D002686:Chitin / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004952:Esters; D047668:Inteins; D015080:Mesna / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D003432:Cross-Linking Reagents; D004952:Esters; D011994:Recombinant Proteins; D002686:Chitin; D003545:Cysteine; D015080:Mesna
keywords: Cell harvesting; Cell lysis; Chitin beads and linking peptide; Chitin-binding domain (CBD); Elution and characterization of protein; Expressed protein ligation (EPL); Peptide