Expression plasmids

The MHAS2168 gene was transferred from pINIT_MHAS2168 to the pACE_C3GH³⁷ and rv1410c gene from pINIT_rv1410c to the pBXC3GH⁵⁶ using FX cloning, resulting in pACE_C3GH_MHAS2168 and pBXC3GH_rv1410c in which the mfs transporters are C-terminally fused to a 3C protease cleavage site, GFP and a His₁₀-tag. Genes of Rv1410 mutants D70N, ΔAB, E147Q, D22N, A411D, and L289R were amplified with primers Rv1410c_for and Rv1410c_rev (Supplementary Table ⁶) using corresponding pFLAG_Rv1410_mutant vectors as templates and then transferred consecutively into pINIT (in which the mutation was confirmed by Sanger sequencing) and pBXC3GH vectors by using FX cloning.

The gene encoding nanobody H2 (Nb_H2) was transferred from pSb_init_Nb_H2 to pBXNPHM3⁵⁷, resulting in pBXNPHM3_Nb_H2 in which the nanobody’s N-terminus is fused to the PelB signal peptide, His₁₀-tag, maltose binding protein and a 3C protease cleavage site. To turn nanobody Nb_H2 into a megabody MB_H2 and Nb_F7 into Mb_F7, the genes encoding Nb_H2 and Nb_F7 and lacking the first thirteen N-terminal residues were transferred to pBXMBQ vector (a kind gift from Eric Geertsma and Benedikt Kuhn), using FX cloning. The resulting constructs pBXMBQ_MB_H2 and pBXMBQ_MB_F7 encode a fusion protein that consists of an N-terminal DsbA signal peptide, the first thirteen N-terminal residues that form the β-strand A of a nanobody, a scaffold protein (HopQ adhesin domain)²² and the rest of the nanobody residues (containing all three CDRs), fused C-terminally to a 3C protease cleavage site, His₁₀-tag, and Myc-tag.