Figure 1.

Construction and production of humanized T‐cell receptors (TCRs) in vitro. (a) The design for different TCR expression cassettes. All these genes were cloned into pET‐28a vector. Vα, variable domain of α chain; Cα, constant domain of α chain; Vβ, variable domain of β chain; Cβ, constant domain of β chain. (b) The gel filtration chromatography of in vitro refolded SRm1 and SRm1g13t eluted with phosphate‐buffered saline. The desired fractions were collected and pooled. (c) The gel filtration chromatography of refolded peptide human leucocyte antigen (pHLA) (MAGE‐A3) and biotinylation efficiency analysis of purified pHLA (MAGE‐A3). The HLA‐A*0201 and β ₂ m inclusion bodies (IBs) were purified firstly, and then denatured protein products were refolded in vitro. The refolded products were purified by AKTA system. The desired fractions were collected and concentrated to do a biotinylation assay, and the reaction efficiency was checked on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Lane M: protein ladder (NEB, P7703V); Lane 1: streptavidin (SA); Lane 2: pHLA (MAGE‐A3) monomer; Lanes 3–5: the mole ratio of SA to pHLA (MAGE‐A3) monomer is 1 : 1, 1 : 4, 1 : 8.