Fig. 4: Site-specific dual incorporation of pcY and Bpa in 7D12.
From: Site-specific encoding of photoactivity and photoreactivity into antibody fragments
a, A single plasmid containing genes to assemble the orthogonal ribosome, our newly evolved Bpa-specific MbPyl(Bpa)RS, AGTA-decoding evolved MbPyltRNAUACU, and 7D12 on orthogonal RBS was constructed. We named this plasmid pSANG-oR-o7D12-Dual-Pyl(Bpa). Cotransformation of this plasmid with pULTRA-pcY allowed for expression of 7D12-32pcY-109Bpa. mRNA, messenger RNA; Pos., position. b, Expression of 7D12-32pcY-109Bpa. Full-length 7D12 observed when expression was performed with both pcY (1 mM) and Bpa (1 mM; +pcY/+Bpa lane). See Extended Data Fig. 7 (some full-length protein also observed for expression performed with only pcY (+pcY/−Bpa lane) that might be due to incorporation of pcY by MbPyl(Bpa)RS in the absence of Bpa). These experiments were repeated twice with similar results. The lane marked L is the Invitrogen SeeBlue Plus2 Pre-stained Protein Standard (catalog no. LC5925). c, ESI-MS of 7D12-32pcY-109Bpa is consistent with site-specific incorporation of pcY and Bpa in 7D12. See Supplementary Fig. 11 for MS data before deconvolution. The ESI-MS data also show a minor peak at 14,537 Da, which is a mass gain of 72 Da on 7D12-32pcY-109Bpa. This peak cannot be explained by dual incorporation of pcY or Bpa, and we are unsure of its origin.
