Anti-GFP (LaG2) is a readily expressible recombinant nanobody which has a high affinity and high specificity against GFP. It was used by us as an extracellular domain of the SynNotch, and accomplished a contact-dependent signal input against GFP. LaG2 fuses two copies of LaG16 using a flexible glycine-rich peptide linker to form a dimerization, ultra-high affinity antibody against GFP[1]. This nanobody recognizes our surEGFP (Part:BBa_K2446051) expressing cells.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]
Biology
Data reported in Fridy PC et al, 2014
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: 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.
As stated by Fridy PC et al: Characteristics of LaG, LaG dimer, and LaM proteins in Friday PC et al, 2014
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.
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.
