Difference between revisions of "Part:BBa K2549003"

Latest revision as of 16:59, 17 October 2018

anti-GFP (LaG16)

Anti-GFP (LaG16) is a readily expressible recombinant nanobody which has a high affinity and high specificity against GFP^[1]. It was used as the extracellular domain of the SynNotch, thus accomplishing the contact-dependent signal input against GFP. Two copies of LaG16 joined to form LaG2 (Part:BBa_K2549002) using a flexible glycine-rich peptide linker, which is a dimerized, ultra-high affinity antibody against GFP. This nanobody recognizes our surEGFP (Part:BBa_K2446051) expressing cells.

Sequence and Features

Biology

Data reported in Fridy PC et al, 2014

Please refer the original article for more details.

We thank the authors carefully documented all the sequences. We used these information, reverse translated and performed codon optimization for better expression in human.

As stated by Fridy PC et al: Overview of nanobody identification and production pipeline. The example nanobody structure shown was obtained from PDB 3K1K. LC-MS/MS, liquid chromatography–tandem mass spectrometry.

As stated by Fridy PC et al: Tandem mass spectra of identified peptides (shown boxed), mapped to the CDR regions of three candidate VHH sequences. The MS-covered regions of these sequences are underlined. Dashed lines indicate overlapping peaks.

As stated by Fridy PC et al: Affinity isolations of yeast Nup84-GFP using the commercial nanobody GFP-Trap, polyclonal anti-GFP or a LaG-16–LaG-2 dimer with a glycine-rich peptide linker. The complex was isolated at various time points, and relative yield was determined by quantification of Coomassie-stained bands of known Nup84-complex components. Data are means from two experiments ± s.e.m.

As stated by Fridy PC et al: Efficacy of LaG nanobodies in immunofluorescence microscopy. (a,b) HeLa cells transiently transfected with tubulin-EmGFP or an EmGFP-tagged mitochondrial marker (in green) were fixed and immunostained with LaG-16 conjugated to Alexa Fluor 568 (AF568, in red). Nuclei were counterstained with DAPI (blue). (c) T. brucei cells expressing EGFP-tagged Sec13 were mixed 1:1 with wild-type cells, fixed and stained with LaG-16–AF568, with DAPI counterstaining.

Fridy PC et al: Characteristics of LaG, LaG dimer, and LaM proteins. Please pay attention to LaG-16, LaG-17, LaG-2.

As stated by Fridy PC et al: Nanobody fluorescent protein binding. SDS-PAGE analysis of high- affinity LaGs that were conjugated to magnetic beads and incubated with various recombinant fluorescent proteins: A. victoria (Av) GFP and its variants CFP, BFP and YFP; a cyan fluorescent protein derived from A. macrodactyla (Am CFP); a yellow fluorescent protein from Phialidium (Phi YFP); and mCherry and DsRed from Discosoma (Ds). Structural models were obtained from PDB 1EMA (Av), PDB 4HE4 (Phi) and PDB 1GGX (Ds); the Am CFP model is a Phyre server prediction. Gels are representative of at least two experiments. Please pay attention to LaG-16, LaG-17, LaG-2.

As stated by Fridy PC et al: Mapping of nanobody binding epitopes on GFP by NMR. Binding epitopes of the 11 highest-affinity nanobodies on GFPuv are shown in three groups according to their location. For each nanobody, two opposite sides of GFPuv are shown (via a 180° rotation along a vertical axis), with the binding site of the respective nanobody colored green. All GFPuv molecules are represented in space-filling mode and have the same orientation in all panels. Maps below Group III in the right column show the GFP-Trap nanobody’s binding epitope (top) and GFPuv’s homodimerization interface (center). For reference, the ribbon diagram at bottom right depicts secondary structure elements of GFPuv, in the same orientation as other panels. Please pay attention to LaG-16, LaG-17, LaG-2.

Note: Part:BBa_K2549002, Part:BBa_K2549003 and Part:BBa_K2549004 have the same Biology section.

References

1. ↑ A robust pipeline for rapid production of versatile nanobody repertoires. Fridy PC, Li Y, Keegan S, ..., Chait BT, Rout MP. Nat Methods, 2014 Dec;11(12):1253-60 PMID: 25362362; DOI: 10.1038/nmeth.3170