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

Aliprandi M, Sparacio E, Pivetta F, Ossolengo G, Maestro R, de Marco A - J. Biomed. Biotechnol. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Protein Chemistry Unit, Cogentech, IFOM-IEO Campus for Oncogenomics, Via Adamello 16, 20139 Milan, Italy.

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.

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Related in: MedlinePlus

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.
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fig3: 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.

Mentions: We next compared the potentiality of the two 3G1B constructs (His- and Fc-tagged) and that of a polyclonal antibody for immunopurifying recombinant SNAP-Twist2-protein from large volumes. The antibodies were covalently bound to sepharose beads and packed into columns. The effective binding between antibodies and beads was verified by analyzing the washing fractions and by boiling a small bead sample for each experimental condition (Figure 3). The target protein was successfully purified from the total lysate by using all three configurations, although a certain yield difference was observed (Figure 3).


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

Aliprandi M, Sparacio E, Pivetta F, Ossolengo G, Maestro R, de Marco A - J. Biomed. Biotechnol. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2963136&req=5

fig3: 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.
Mentions: We next compared the potentiality of the two 3G1B constructs (His- and Fc-tagged) and that of a polyclonal antibody for immunopurifying recombinant SNAP-Twist2-protein from large volumes. The antibodies were covalently bound to sepharose beads and packed into columns. The effective binding between antibodies and beads was verified by analyzing the washing fractions and by boiling a small bead sample for each experimental condition (Figure 3). The target protein was successfully purified from the total lysate by using all three configurations, although a certain yield difference was observed (Figure 3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Protein Chemistry Unit, Cogentech, IFOM-IEO Campus for Oncogenomics, Via Adamello 16, 20139 Milan, Italy.

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.

Show MeSH
Related in: MedlinePlus