Peptide-binding Nanobody for Biochemical Applications
Inventors: Hidde Ploegh, Jingjing Ling, Ross Cheloha
Invention Types: Research Tool
Keywords: Antibody, Method of Use, Protein, Reagent, Molecular biology, ImagingFor More Information Contact: Ives, Catherine L.
Epitope tags are useful tools in molecular biology to identify, purify, and quantify a protein of interest when a specific and high-affinity antibody cannot be generated. Biologists therefore often tag their proteins of interest with epitope tags where a good antibody has already been developed, for example the hemagglutinin (HA)-tag derived from influenza hemagglutinin and the Myc-tag derived from c-Myc transcription factor. Nanobodies, also known as VHHs, are Camelid-derived single-domain antibody fragments with many advantages over conventional antibodies. While many VHHs have been identified, few have been shown to recognize peptide epitopes, and even fewer have been used as tool for epitope tagging applications.
The Ploegh lab has identified a nanobody, VHH05, that recognizes a 14 amino acid peptide fragment of the E2 ubiquitin-conjugating enzyme UBC6e (“6e”), with low nanomolar affinity. As proof-of-principle, the group tested the nanobody-peptide system with a 6e-tagged EGFP in a variety of applications. VHH05 efficiently and specifically detected the 6e-tagged EGFP in ELISA and sandwich ELISA; furthermore, EGFP-6e expressed in mammalian cells was detected by VHH05 in immunoblot and FACS, and was cleanly immunoprecipitated. VHH05 can be used to detect and quantify the tagged protein after immunoprecipitation or in conjunction with conventional antibodies for co-immunoprecipitated targets.
The most powerful benefit of VHHs over conventional antibodies is that VHHs retain their specificity and strong binding capacity in the reducing environments of the cytosol because they do not require disulfide bonds for activity. Appropriately designed constructs of VHH05 can be tethered to the ER or mitochondria, which then recruits 6e-tagged EGFP to these locations. This directing system allows analysis of protein functions at specific locations within the cell.
• A kit for molecular biology applications such as protein purification and protein detection (ELISA, western blotting, immunostaining, etc).
• A kit for cellular biology applications such as directing trafficking or localization of a protein of interest, tethering proteins, or cellular imaging.
• Cytosolic expression: VHHs do not require disulfide bonds to maintain their structure, unlike traditional antibodies, and thus can be expressed and retain target specificity in the reducing environment of the cytosol.
• Low cost: High expression yield in low-cost E. coli help to drive down costs compared to conventional antibodies.
• Small size: VHHs are highly tissue-permeable and cause less offset between label and target, for superior in vitro and in vivo imaging.
License, sponsored research, collaboration
Key Publications: Ling J., Cheloha R.W., McCaul N., Sun Z.J., Wagner G, Ploegh H.L. Modulation of the E2 ubiquitin-conjugating enzyme UBC6e with a camelid single domain antibody fragment identifies a region critical for its activity. Under Review J Biol Chem.
IPStatus: Pat. Pend.