Expanding the Structural Diversity of Protein Building Blocks with Noncanonical Amino Acids Biosynthesized from Aromatic Thiols

Abstract

Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins is valuable for both scientific and biomedical applications.Toexpand the structural diversity of available ncAAs and to reduce the burden of chemically synthesizing them, we have developed ageneral and simple biosynthetic method for genetically encoding novel ncAAs into recombinant proteins by feeding cells with economical commercially available or synthetically accessible aromatic thiols.W ed emonstrate that nearly 50 ncAAs with ad iverse arrayo fs tructures can be biosynthesized from these simple small-molecule precursors by hijacking the cysteine biosynthetic enzymes,a nd the resulting ncAAs can subsequently be incorporated into proteins via an expanded genetic code.M oreover,w ed emonstrate that bioorthogonal reactive groups such as aromatic azides and aromatic ketones can be incorporated into green fluorescent protein or at herapeutic antibody with high yields,a llowing for subsequent chemical conjugation.

Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

“…In summary, in this study, we generated 28 Mb PylRS variants which can incorporate 23 ncAAs and one cAA. To the best of our knowledge, 17 of these ncAAs (besides 1, 3, 4, 17, 27, and 28) were not ribosomally incorporated by amber suppression before and 20 of them were not previously incorporated with the PylRS system [ 33 , 35 , 40 , 41 ].…”

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

“…60 Recently there have been a number of biosynthetic pathways adapted for GCE to overcome this bottleneck, though many still rely on supplementation of precursor molecules not endogenous to the expression host. 60, 61 NhpSer biosynthesis begins from phosphoenolpyruvate, and so PermaPhos Ser expression is functional in standard complex media (e.g. 2xYT and Terrific Broth).…”

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

“…5, PDB ID "6DN0"). The protein was produced and purified from the periplasmic space 41,42 , in E. coli BL21 (DE3) cotransformed with pULTRA-pTAFRS and pET22b-HER2-scFv-S9/K42/ V15TAG plasmids. A HER2-scFv-K42 pTAF mutant, which had a high expression yield (30 mg/L), was chosen for further characterization; and the mutant and wild-type proteins were confirmed by SDS-PAGE and high-resolution mass spectrometry (Supplementary Figs.…”

“…It would have the potential for enhanced tumor penetration and better in vivo pharmacokinetics. J591 Fab containing a A121 pTAF mutation, where the alanine at 121 position was mutated to ncAA pTAF by sitedirected mutagenesis of alanine codon to an amber codon, was purified from the periplasmic space in E.coli BL21(DE3) along with the wild- type protein 41 . A121 site was chosen on the basis of its surface accessibility, high expression yield and high conjugation efficiency 10 .…”