Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry
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NP_20130312_Click_MDW[1].pdf
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Author(s)
Witte, Martin D.
Theile, Christopher S.
Wu, Tongfei
Guimaraes, Carla P.
Blom, Annet E. M.
Ploegh, Hidde
Date Issued
August 2013
Journal
Nature Protocols
Publisher
Nature Publishing Group
Citation
Witte, Martin D, Christopher S Theile, Tongfei Wu, Carla P Guimaraes, Annet E M Blom, and Hidde L Ploegh. “Production of Unnaturally Linked Chimeric Proteins Using a Combination of Sortase-Catalyzed Transpeptidation and Click Chemistry.” Nat Protoc 8, no. 9 (August 29, 2013): 1808–1819.
Version
Original manuscript
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Whitehead Institute for Biomedical Research
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1038/nprot.2013.103
