2.2. Soluble Expression of TCRs and aCD3 in HEK293 Cells

The sequences of the TCRs were obtained from previous studies [21,23]. Fc fused TCR genes (tagged with sortase A recognition sequence) were synthesized using the Sangon Biotech (Shanghai) Co., Ltd. and then inserted into the pMH3 plasmid (AmProtein China Cooperation Ltd., Hangzhou, China) to construct the expression plasmid. HEK293 cells in the logarithmic growth phase were transfected with the expression plasmids and cultured for 4 days according to the method mentioned in the cell culture. The supernatant of the HEK293 cells was then collected and the TCRs were purified using protein A column (HiTrap^TM Protein A HP, GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, USA). The purified TCRs were analyzed using SDS-PAGE in denatured condition after being de-glycosylated using PNGase F (Sigma-Aldrich, St. Louis, MO, USA).

The sequences of the variable domain of the aCD3 were obtained from a previous study [24]. The aCD3 genes (Fab, tagged with (G₄S)₃-LPETG-6×His at the C-terminus of the heavy chain) were synthesized using the Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) and were then inserted into the pMH3 plasmid to construct the expression plasmid. The aCD3 was expressed using HEK293 cells according to the method mentioned for TCR expression and purified using Ni column (HisTrap^TM HP, GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK).