Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry

Martin D. Witte, Christopher S. Theile, Tongfei Wu, Carla P. Guimaraes, Annet E. M. Blom, Hidde L. Ploegh*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

57 Citaten (Scopus)

Samenvatting

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-alpha, 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.

Originele taal-2English
Pagina's (van-tot)1808-1819
Aantal pagina's12
TijdschriftNature protocols
Volume8
Nummer van het tijdschrift9
DOI's
StatusPublished - sep.-2013
Extern gepubliceerdJa

Citeer dit