Anti-GCN4 single-chain variable fragment fused to superfolder GFP, a GB1 solubility tag and VP64 (scFv-GCN4-sfGFP-GB1-VP64)
Antibody half of the SunTag system. A single-chain variable fragment (scFv) against the GCN4 peptide, fused to superfolder GFP, the GB1 solubility tag and the VP64 activation domain. Each copy binds one GCN4 peptide in a SunTag array, so one array recruits many VP64 domains to a promoter.
Length: 2031 bp (677 aa)
Type: Single-chain antibody fragment fusion
Function: Binds GCN4 peptide epitopes and delivers a fused effector domain in multiple copies to the protein carrying the array
Origin: Anti-GCN4 scFv fused to sfGFP, GB1 and VP64
Characteristics
An scFv joins the epitope-binding regions of an antibody light and heavy chain into one polypeptide, which folds in the cytoplasm where whole antibodies do not. The bare scFv-GCN4-GFP still aggregates in human cells at high expression, and fusing superfolder GFP alone, or together with the small GB1 solubility tag, to the C terminus of the GCN4 antibody almost eliminates aggregation even at high expression. By FRAP on a mitochondria-tethered GCN4 array, the antibody binds the v4 peptide array in vivo and dissociates with a slow off rate. A nuclear localization signal is often added to sequester unbound antibody in the nucleus.
Applications: Effector-delivery half of dCas9-SunTag gene activation, coexpressed with a dCas9-GCN4 array to activate CXCR4 and CDKN1B in K562 cells. Without VP64 the same antibody carries sfGFP for signal amplification, making single protein molecules and genomic loci bright enough for long-term imaging; single SunTag foci average 18-fold brighter than single sfGFP molecules.
Limitations: Functions only with a coexpressed GCN4 peptide array, so it is never used alone. Unbound antibody raises background and a nuclear localization signal is normally added to sequester it. The bare scFv-GCN4-GFP aggregates at high expression levels without the sfGFP and GB1 solubility fusions.
Modifications: sfGFP and GB1 solubility fusions at the scFv C terminus; VP64 activation domain; nuclear localization signal in the activator form
