Preparation of unnatural N-to-N and C-to-C protein fusions - PubMed (original) (raw)
Preparation of unnatural N-to-N and C-to-C protein fusions
Martin D Witte et al. Proc Natl Acad Sci U S A. 2012.
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.
Conflict of interest statement
Conflict of interest statement: The authors declare that a patent application on the production of N-to-N and C-to-C fusions has been filed.
Figures
Fig. 1.
Schematic overview of the approach.
Fig. 2.
Proof of concept for the synthesis of N-to-N fused proteins. (A) structures of the used N-terminal probes 1 and 2. (B and C) Labeling of his-tagged UCHL3 with dimeric UbVME. (B) Coomassie brilliant-blue–stained Tris-tricine gel. (C) Immunoblot using anti-His antibody. Ub-UbVME*, ubiquitin-UbVME bound to a single UCHL3; UbVME2*, dimeric UbVME bound to a single UCHL3 molecule; UbVME2**, dimeric UbVME bound to two UCHL3 molecules.
Fig. 3.
C-to-C homodimeric antibodies. (A) Structures of probes 3 and 4. (B) Dimerization of anti-GFP. (C) Size exclusion experiment demonstrating that both anti-GFPs bind GFP. From Top to Bottom: anti-GFP dimer; GFP; anti-GFP dimer + 0.4 equiv GFP; anti-GFP dimer + 1.6 equiv GFP; anti-GFP dimer + 4.8 equiv GFP (excess).
Fig. 4.
(A) FACs staining of mouse lymph node cells with anti-MHC II–anti-GFP antibodies. (Upper) Staining observed in wild-type cells. (Lower) Staining of MHC class II-deficient cells. (B) In vivo delivery of GFP. Mice were injected with 50 μg bispecific antibodies and either received directly intraperitoneally or after 1 h i.v. 50 μg GFP. Stained cells were analyzed by flow cytometry.
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