Preparation of unnatural N-to-N and C-to-C protein fusions.
Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein-protein fusions, the method reported can be extended to connecting proteins with any entity of interest.
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Martin D
MD
Witte
Juan J
JJ
Cragnolini
Stephanie K
SK
Dougan
Nicholas C
NC
Yoder
Maximilian W
MW
Popp
Hidde L
HL
Ploegh
D019881:Aminoacyltransferases / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002244:Carbon / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008956:Models, Chemical; D015394:Molecular Structure; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000493:pharmacokinetics; D009584:Nitrogen / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization
D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D049452:Green Fluorescent Proteins; D002244:Carbon; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D009584:Nitrogen