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.

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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.

Fig. 1.

Schematic overview of the approach.

Fig. 2.

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.

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.

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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