FIGURE 7.
Effects of replacement of Asn residues in anti-hCG VHH. A, Asn residues 52, 74, and 84 in anti-hCG VHH-H14 were substituted by Ser, Ser, and Thr, respectively, resulting in the N52S/N74S/N84T mutant. B, N52S/N74S/N84T mutant (100 nm) was treated with heating (90 °C for 5 min)-cooling (20 °C for 5 min) cycles (black closed box) or continuous incubation at 90 °C followed by refolding at 20 °C for 5 min (black open box). The theoretical curve for N52S/N74S/N84T (solid line) was fitted using 0.0025 min−1 as a first-order kinetic constant kden. From this kden, the half-life of the N52S/N74S/N84T mutant was calculated to be 274 min (m). Data for wild-type VHH treated with continuous incubation and its theoretical curve are presented as red circles and broken line, respectively. The SPR signals for untreated wild-type (red broken line) and N52S/N74S/N84T (black solid line) VHHs (100 nm) are presented in the inset. In all panels, wild-type and N52S/N74S/N84T mutant VHHs are abbreviated as WT and Mutant, respectively. Error bars represent S.D. CDR, complementarity determining region.