2.2. Protein Expression Constructs

Expression constructs are based on the pcDNA™5/FRT/TO vector (Thermo Fisher, Waltham, MA, USA). Reference constructs encode a minimal version of the tissue plasminogen activator (mini-tPA) signal sequence (MDAMKRGLCCVLLLCGAVFVSPSAA) followed by the Env sequence and a C-terminal hexahistidine-tag (GS-H₆). Variants optimized for cysteine-mediated coupling encode the mini-tPA signal sequence followed by the coupling tag (CAAC), a glycine-serine-linker ((G₄S)₃), the Env sequence and a C-terminal hexahistidine-tag (GS-H₆). SOSIP-constructs encode the two cysteines for the formation of the SOS disulfide bridge (A501C, T605C) the IP exchange (I559P) and the optimized Furin protease cleavage site (R₆) as described earlier [6]. The native flexible linker (NFL) constructs contain the same amino acid exchanges but the SOS disulfide bridge is omitted and the Furin protease cleavage site is substituted by a flexible linker ((G₄S)₂) as described before [28]. Env sequences were the stabilized BG505 sequence including the glycan-knock-in (T332N) to complete the 2G12-bnAb epitope [6] and a stabilized Clade C consensus sequence which bears the G473T exchange which eliminates CD4 binding [29]. This stabilized ConC trimer (originally referred to as ConCv5 KIKO) is the result of a stepwise stabilization process [30]. For simplicity, we refer to this Env trimer in this study just as ConC-SOSIP and ConC-NFL. As a control, a variant without an N-terminal tag was generated by deletion of the coupling tag and the N-terminal glycine-serine-linker. The plasmid encoding the furin gene was used as described earlier [27]. All constructs were optimized for human codon usage.