Figure 9.

Binding properties and thermostability of parental T1 TCR and the F45βY interface mutant. A, T1 and F45βY were cloned with N-terminal His₆ tag in pET28a for expression in E. coli. F45βY contained additional C-terminal Avitag sequence, hence leading to slight difference in mobility on a 4–20% SDS-PAGE (inset). Binding of each protein to MART-1·HLA-A2 was assessed by detecting the β2m subunit of HLA-A2 by ELISA. B and C, kinetic titration to determine dissociation constants of soluble T1 (B) and interface mutant F45βY (C) toward MART-1·HLA-A2 by surface plasmon resonance. Biotinylated MART-1·HLA-A2 was coated on a SPR sensor chip, followed by injection of soluble TCRs over the coated surface. The association and dissociation phases were monitored for 2 and 3 min, respectively. Resulting response was plotted against time, and data were fitted and analyzed using Biaevaluation version 4.1 software. D, DSF profiles of soluble scTCRs (mixed with SyproOrange) when exposed to increasing temperature. Melting temperature can be determined as temperature where half-maximal increase in fluorescence is noted. First derivative curves of DSF data (temperature (T) versus derivative (∂F/∂T)) to calculate precise T_m values are also shown (inset). E, thermostability of yeast-displayed scTCRs was assessed by exposing yeast cells to temperatures ranging from 30 to 80 °C for 30 min, followed by staining with saturating concentration of monomeric MART-1·HLA-A2. The percentage decrease in binding (MFU) was calculated relative to binding at 30 °C and plotted against temperature to obtain T_m.