1. Volume 25, Issue 10, Pages 1611-1622.e4 (October 2017)
Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability 
Takao Arimori, Yu Kitago, Masataka Umitsu, Yuki Fujii, Ryoko Asaki, Keiko Tamura-Kawakami, Junichi Takagi 
Structure 
Volume 25, Issue 10, Pages e4 (October 2017)
DOI: /j.str
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2. Structure 2017 25, 1611-1622.e4DOI: (10.1016/j.str.2017.08.011)
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3. Figure 1 Fusion of the Antibody Fv Region with the SARAH Domain
(A) Diagrams of domain architecture for antibody fragments and design concept of Fv-clasp. Each Ig fold β sandwich domain is represented by an oval, with the domain linkers shown in either solid or broken lines. Homodimeric SARAH domain structure (shown for human Mst1, PDB: 2JO8) is topologically compatible with the fusion at the Fv C termini, leading to the design of “Fv-clasp.”
(B) General design of Fv-clasp constructs. Heavy (VH1-113) and light (VL1-108) chains of a given antibody are individually fused with a 49-residue SARAH domain via a two-residue (Gly-Ser) linker. Sequences for five different SARAH domains are shown. Two Cys residues in the hRaf1 SARAH domain were substituted with Ser (red) to avoid undesired disulfide bond formation. Residues 24 and 35, which are mutated to Cys to form an asymmetric inter-chain disulfide bond based on the homodimeric Mst1 structure, are shown with a black background.
(C and D) Expression of anti-C8 peptide-antibody P20.1 fragments. Various lengths of P20.1 heavy- and light-chain constructs were co-transfected into Expi293F cells, and the culture media were immunoprecipitated with C8-immobilized beads followed by SDS-PAGE under non-reducing condition and Oriole staining. Results with Fv-clasps containing unmodified (C) or Cys-substituted (D) SARAH domains are shown.
(E and F) Purification of bacterially expressed Fv-clasp(v1) after refolding. Shown are (E) size-exclusion chromatography (SEC) and (F) anion-exchange chromatography (AEC) profiles during the preparation of 12CA5 Fv-clasp(v1), corresponding to steps 3 and 4 in Figure S1, respectively. On the right, SDS-PAGE profiles for the major peak in (E) (asterisk) or (F) (labeled A–C) are shown. Irrelevant lanes were eliminated from the gels.
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4. Figure 2
The Fv-clasp(v1) Folds into a Disulfide-Bonded Two-Domain Protein as Designed
(A and B) Crystal structures of P20.1 Fv-clasp(v1) (A) and 12CA5 Fv-clasp(v1) (B) are shown as cartoon presentation with the VH-SARAH and VL-SARAH colored in green and cyan, respectively. The bound antigen peptides are shown as stick models in the CPK color scheme. The SDS-PAGE profiles of the purified samples under reducing (R) and non-reducing (NR) conditions are shown in the left.
(C) Comparison between the crystal structures of P20.1 Fv-clasp(v1) (green and cyan) and P20.1 Fab (orange, PDB: 2ZPK), superposed at the Fv region. The expanded view of the peptide-antibody interface is shown in the right. Note that the configuration of the C8 peptide, side-chain conformation of antibody residues involved in the interaction, and position of the water molecule mediating the inter-molecular hydrogen bond are all completely conserved between the two structures.
(D and E) Recognition of HA-tag peptide by 12CA5. In (D), a translucent surface model for the 12CA5 Fv-clasp(v1) is shown with the bound HA peptide (CPK colored stick model), of which all nine residues could be assigned in the electron density (Fo − Fc omit map contoured at 2.5 σ level). (E) 12CA5 CDR loop residues making direct contact with the HA peptide are shown in stick models and viewed from the same orientation as (D).
(F) Inter-domain mobility between the Fv and the SARAH domain. Four crystallographically independent Fv-clasp(v1) structures are superposed either at the Fv region (left) or the SARAH domain (right), and shown as cartoon presentation with the indicated colors, except for the superposed segment in light gray. See also Figure S2.
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5. Figure 3 Structure-Guided Redesigning of the Fv-clasp
(A) Introduction of the inter-chain disulfide bond. The schematics depict the design strategy to make Fv-clasp(v2), by changing the position of the engineered inter-chain disulfide (black line connecting dots). Below the cartoon is a close-up view of the crystal structure of P20.1 Fv-clasp(v1) (crystal form-1) near the designed inter-chain disulfide bond.
(B) Heterodimer formation of the newly designed Fv-clasp(v2). Two differently Cys-substituted (i.e., v1 and v2) and C-terminally His-tagged Fv-clasp constructs for P20.1 and TS2/16 antibodies were expressed in Expi293F cells, and the culture supernatants were subjected to pull-down assay using Ni-NTA agarose followed by SDS-PAGE and Coomassie staining.
(C) Thermal stability of the Fv-clasp proteins. Thermal denaturation curves measured by SYPRO orange fluorescence during the temperature scan from 25°C to 85°C are shown for various fragment formats (scFv, gray; Fv-clasp(v1), blue; Fv-clasp(v2), red) of indicated antibodies. The –dF/dT peak positions represent Tm values and are summarized in Table S1. See also Figure S3.
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6. Figure 4 Improved Crystallizability of the Fv-clasp(v2)
(A–D) Determined crystal structures of 12CA5 Fv-clasp(v2) (A), NZ-1 Fv-clasp(v2) (B), TS2/16 Fv-clasp(v2) (C), and P20.1 Fv-clasp(v2) (D) are shown as differently colored cartoon presentations, along with the photographs of the crystals used in the data acquisition. The introduced disulfide bonds and the bound antigen peptides, where applicable, are shown as stick models. Resolution of each structure is also indicated.
(E) Superposition of the four Fv-clasp(v2) structures. Heavy (light green) and light (light cyan) chains are shown in cartoon presentation, with the three hydrophobic residues (11(H), 40(L), and 83(L)), at the bottom of the Fv domain highlighted in red sphere models. Note that the Fv-clasp(v2) structures exhibit much less inter-subdomain mobility compared with the Fv-clasp(v1) (Figure 2F). See also Figures S4 and S5.
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7. Figure 5
Use of Fv-clasp(v2) in the “Crystallization Chaperon” Applications
Crystallization and structure determination of the sorLA Vps10p domain in complex with the propeptide ligand (A and C) as well as the α6β1 headpiece fragment (B and D) are assisted by complexation with the Fv-clasp(v2) of respective antibodies. (A) Structure of sorLA Vps10p (orange) bound by its propeptide (light blue), and Fv-clasp(v2) of antibody (gray) are shown in cartoon presentation. (B) Structure of the α6β1 headpiece in complex with TS2/16 Fv-clasp(v2). α6 and β1 subunits of integrin are shown in light blue and orange cartoon models, respectively. In both (A and B), photographs of the crystals used for the diffraction experiments are shown as insets. Scale bars, 100 μm. (C and D) Packing arrangements in the crystal. Antigen molecules are shown in dark- and light-gray surface models, while Fv-clasp(v2) molecules are shown in dark and light magenta.
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