The availability of a recombinant anti-SNAP antibody in VHH format amplifies the application flexibility of SNAP-tagged proteins

Abstract

Antibodies are indispensable reagents in basic research, and those raised against tags constitute a useful tool for the evaluation of the biochemistry and biology of novel proteins. In this paper, we describe the isolation and characterization of a single-domain recombinant antibody (VHH) specific for the SNAP-tag, using Twist2 as a test-protein. The antibody was efficient in western blot, immunoprecipitation, immunopurification, and immunofluorescence. The sequence corresponding to the anti-SNAP has been subcloned for large-scale expression in vectors that allow its fusion to either a 6xHis-tag or the Fc domain of rabbit IgG2 taking advantage of a new plasmid that was specifically designed for VHH antibodies. The two different fusion antibodies were compared in immunopurification and immunofluorescence experiments, and the recombinant protein SNAP-Twist2 was accurately identified by the anti-SNAP Fc-VHH construct in the nuclear/nucleolar subcellular compartment. Furthermore, such localization was confirmed by direct Twist2 identification by means of anti-Twisit2 VHH antibodies recovered after panning of the same naïve phage display library used to isolate the anti-SNAP binders. Our successful localization of Twist2 protein using the SNAP-tag-based approach and the anti-Twist2-specific recombinant single-domain antibodies opens new research possibilities in this field.

Figure 1

Application of anti-SNAP antibodies in western blot experiments. (a) Detection of 100 and 10 ng of blotted purified recombinant GST-SNAP protein by means of the VHH antibodies 3C4B and 3G1B. (b) Detection of overexpressed GST-SNAP in bacterial total lysates using the same VHH antibodies. The secondary antibody anti-llama immunoglobulins used for detection recognized nonspecifically a further band of 32 kDa.

Figure 2

Application of anti-SNAP antibodies in immunopurification experiments. (a) Purified GST-SNAP (0.5 or 0.1 μg) were incubated in the presence of the biotinylated anti-SNAP VHH 3G1B (5 or 1 μg), and the antigen-antibody complex was precipitated using avidin beads. The proteins were separated by SDS-PAGE, blotted, and finally detected using either anti-GST (upper membrane strip) or anti-VHH specific antibodies (lower membrane strip). Biotinylated VHHs were incubated in the presence of avidin beads and in the absence of GST-SNAP as a control of the pull-down efficiency. (b) Lysates from mammalian HEK293T cells transiently transfected with 20 μg/plate plasmid for expressing the SNAP-Twist2 fusion construct were incubated with the anti-SNAP 3G1B-Fc(R) antibody, and the antigen-antibody complex was precipitated by means of Protein A.

Figure 3

Comparison of the protein yields obtained by immunopurification using different antibody formats. The protein SNAP-Twist2 was immunopurified from total lysate using sepharose columns to which the following antibodies were covalently linked: llama IgG anti-SNAP polyclonal antibodies; 6xHis tagged llama recombinant antibody 3G1B in VHH format; the same 3G1B antibody fused to the rabbit Fc domain.

Figure 4

Double staining of SNAP-tagged Twist2 obtained by coupling specific antibodies and benzylguanine-fluorochromes. HeLa cells were transiently transfected using 20 μg/plate of plasmid for the expression of the SNAP-Twist2 protein. Its subcellular localization was identified: (a) and (e) by the in vivo addition of the permeable SNAP-Cell TMR Star fluorochrome (dilution 1 : 200); (b) by means of the purified recombinant anti-SNAP antibody 3G1B fused to the rabbit Fc (5 μg/mL); (f) by means of the cell culture supernatant containing the recombinant anti-Twist2 VHH antibody 2C12 (dilution 1 : 2); (d) and (h) are the merged images of (a)+(b) and (e) + (f), respectively; (c) and (g) correspond to the nuclear DAPI staining; (i), (j), and (k) are nontransfected cells (controls) treated with the indicated antibodies. HeLa cells transfected with SNAP-Epsin8 were stained with SNAP-Cell TMR Star fluorochrome (dilution 1 : 200) to rule out a SNAP-dependent nuclear staining (l).