Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability

Abstract

Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6β1.

AMPA receptor anchoring at CA1 synapses is determined by an interplay of N-terminal domain and TARP γ8 interactions

“…GluA1 and GluA2Q both with and without their NTD were tagged at their N-termini with an HA-epitope, which can be labelled live using a fluorescently-labelled anti-HA single-chain Fv-fragment (scFv-Clasp) (Fig. 4a) 37 . This small, monovalent probe (~5 nm diameter) prevents receptor clustering by epitope cross-linking and increases localisation precision in single-molecule imaging.…”

Site-specific epitope insertion into recombinant proteins using the MAP tag system

“…In order to facilitate the crystallization of PMab-1, we first converted it to a small antibody fragment format ‘Fv-clasp’ developed recently ( 12 ). Fv-clasp is a fusion of an anti-parallel coiled-coil structure derived from the human Mst1 SARAH domain to the antibody variable region ( i.e.…”

“…Plasmids for bacterial expression of PMab-1 in the Fv-clasp(v2) format (non-tagged version), i.e. PMab-1V H (S112C)-SARAH and PMab-1V L -SARAH(S37C), were prepared as previously described using cDNA clones of rat anti-MAP monoclonal antibody PMab-1 (IgG 2a , κ) isolated previously ( 12 , 13 ). N43Q and N54Q mutations were introduced in the V H domain of the PMab-1V H (S112C)-SARAH construct by a QuikChange mutagenesis, and the resultant plasmid was used for production of the PMab-1(QQ) Fv-clasp mutant in combination with the PMab-1V L -SARAH(S37C).…”

“…N43Q and N54Q mutations were introduced in the V H domain of the PMab-1V H (S112C)-SARAH construct by a QuikChange mutagenesis, and the resultant plasmid was used for production of the PMab-1(QQ) Fv-clasp mutant in combination with the PMab-1V L -SARAH(S37C). Expression and purification of the Fv-clasp samples were carried out essentially according to the previously established sample preparation protocol ( 12 ), but with minor modifications. Namely, the denatured samples after the cell lysis by sonication were diluted 20-fold in modified refolding buffer (50 mM MES, 9.6 mM NaCl, 0.4 mM KCl, 2 mM MgCl 2 , 0.5 M L-arginine, 1 mM reduced glutathione and 0.1 mM oxidized glutathione) in one step.…”

“…Here, we conducted a structural analysis of PMab-1 complexed with the MAP tag peptide by utilizing a hyper-crystallizable ‘Fv-clasp’ format developed recently in our group ( 12 ). The 2.49 Å resolution crystal structure of PMab-1 Fv-clasp revealed recognition mechanism of MAP tag peptide, suggesting that the tag sequence can be shortened to 8 residues from its original 14 residues and is compatible with the insertion into loop regions.…”