Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera

Abstract

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.

Direct selection of functional fluorescent-protein antibody fusions by yeast display

“…The scTGP clones p1-1 [10], 14C2 [12,39] and Z3 [12] were constructed using circular polymerase extension cloning (CPEC) assembly [40] from yeast display (pDNL6) clones that contained the corresponding scFv gene [32]. Briefly, the scFv genes and the vector sequences were amplified by inverse PCR and the TGP sequence was amplified using standard PCR with primers containing overlapping sequences that facilitated CPEC assembly.…”

“…Consequently, many published chimeras have very low expression levels [20], a problem that has been addressed with the split GFP system [27], in which a short tag derived from GFP, rather than GFP itself, is fused to a scFv [28], or by using particularly stable scFvs [22] or nanobodies [29]. An unexpected partial solution to this problem was the finding that fluorescent proteins could be used as linkers between VH and VL [30,31], with the resultant chimeras often better expressed than the parental scFv [32]. We named these chimeric proteins scFPs (single chain fluorescent proteins) [32], according to the fluorescent protein used-scTGP, when thermal green protein [33] was used, and scGFP, when superfolder GFP [34] was used.…”

“…An unexpected partial solution to this problem was the finding that fluorescent proteins could be used as linkers between VH and VL [30,31], with the resultant chimeras often better expressed than the parental scFv [32]. We named these chimeric proteins scFPs (single chain fluorescent proteins) [32], according to the fluorescent protein used-scTGP, when thermal green protein [33] was used, and scGFP, when superfolder GFP [34] was used. Unfortunately, while some poorly expressed scFvs were expressed far better as scFPs, not all scFPs were equally well-expressed.…”

Engineering a Yellow Thermostable Fluorescent Protein by Rational Design

“…Thermo green protein (TGP) (RCSB PDB 4TZA) is an unusually thermostable and non-aggregation-prone fluorescent protein that was engineered from the fluorescent protein eCGP123 . The fluorescent protein eCGP123 was derived from the synthetic consensus green protein (CGP) with directed evolution used to improve thermostability. , TGP has strong advantages over other available FPs in experiments utilizing harsh thermophilic conditions or when there are concerns about protein aggregation affecting assay results, for example, in amyloid assays. ,, TGP has also been used to construct a chimera between the light and heavy chain variable regions of antibodies, permitting one-step fluorescence assay for fluorescent-activated cell sorting . TGP has an 87% identity to monomeric Azami Green (mAG) from Galaxea fascicularis and 33.3% identity to the Aequorea victoria GFP.…”

“… 6 , 11 , 12 TGP has also been used to construct a chimera between the light and heavy chain variable regions of antibodies, permitting one-step fluorescence assay for fluorescent-activated cell sorting. 13 TGP has an 87% identity to monomeric Azami Green (mAG) from Galaxea fascicularis and 33.3% identity to the Aequorea victoria GFP. Like other fluorescent proteins, TGP is an 11-stranded β barrel protein with a central α helix that contains three residues (QGY) that form the chromophore.…”