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Preparation of unnatural N-to-N and C-to-C protein fusions.

ABSTRACT: 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.

SUBMITTER: Witte MD 

PROVIDER: S-EPMC3409725 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Preparation of unnatural N-to-N and C-to-C protein fusions.

Witte Martin D MD   Cragnolini Juan J JJ   Dougan Stephanie K SK   Yoder Nicholas C NC   Popp Maximilian W MW   Ploegh Hidde L HL  

Proceedings of the National Academy of Sciences of the United States of America 20120709 30

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 i  ...[more]