Fig. 6: The ncAAs biosynthetic pathway applied for intracellular synthesis of antibody fragments.

a Depiction of plasmids for ncAAs synthesis and incorporation into antibody fragments. Plasmids that expression of CsLTA and RpTD for ncAAs cascade catalysis and pAzFRS (2×)/tRNA^Tyr pairs were same as Fig. 5a. Mutants of four antibody fragments were constructed in the pET22b vector. pAzFRS (2×) was controlled by arabinose-controlled pBAD promoter, tRNA was controlled by constitutive promoter and other genes were expressed under IPTG-induction. b The yields of antibody fragments containing ncAAs (Mutant) after purification and comparison with wild type (WT). The cell was cultured with 1 mM pAzBzH, 50 mM Gly and 20 μM PLP and induced by arabinose and IPTG for 24 h. The proteins were purified with Ni-NTA column. c High-resolution mass of purified antibody fragments with pAzF (The high-resolution mass spectrometry of wild type antibody fragments were shown in Supplementary Fig. 25). The purification experiments were repeated one time with result. d Confocal images of Her2-positive SK-Br-3 cells and Her2-negative MDA-MB-468 cells stained with anti-Her2-Fab-A121pAzF-AF488 conjugate. In consistent with literature (Ref.³⁵), Her2-negative MDA-MB-468 cells was exploited as negative control in the fluorescence assay rather than wild type antibody because the click reaction between azide and alkyne group is commonly known as classic biorthogonal chemistry. Scale bars = 10 µm. Assays were repeated one time with result. Source data of (b) are provided in Source Data file.