title: Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry.
abstract: Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Theile
authors.forename: Tongfei
authors.initials: T
authors.lastname: Wu
authors.forename: Carla P
authors.initials: CP
authors.lastname: Guimaraes
authors.forename: Annet E M
authors.initials: AE
authors.lastname: Blom
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D057930:Click Chemistry / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D010446:Peptide Fragments / Q000096:biosynthesis; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D002244:Carbon; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D009584:Nitrogen