A noncanonical amino acid-based relay system for site-specific protein labeling

Abstract

Genetically site-specific introduction of noncanonical amino acids (ncAAs) for protein conjugation generally requires incorporation through exogenous feeding of chemically synthesized ncAAs. We developed a p-amino-phenylalanine (pAF)-based relay system that enables site-specific functionalization of proteins without chemical synthesis of the building blocks. pAF was biosynthesized under optimized conditions, followed by site-specific incorporation into a specific protein residue. The resulting protein was ready for functionalization using an oxidative conjugation reaction. We demonstrated the use of this relay system by preparing a fluorophore-labeled anti-HER2 single-chain variable fragment antibody for fluorescent imaging.

Addition of Isocyanide-Containing Amino Acids to the Genetic Code for Protein Labeling and Activation

“…Escherichia coli BL21 (DE3) cells containing the pET22b-T5-sfGFP* plasmid were independently transformed with each of the PylRS mutants, encoded on plasmid pUltra-PylRS. 60,61 A quantitative fluorescence assay was carried out in LB medium in the presence or absence of 1 mM NCibK or NCK. We identified a PylRS mutant (HibK-1) containing two amino acid substitutions (Cys313Thr and Tyr349Phe) that exhibited an increase in fluorescence in the presence of NCibK, suggesting successful expression of full-length NCibK-containing sfGFP ( Figure S1).…”

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

“…Thiols 7, 21,and 22 contain commonly used bioorthogonal reactive groups for producing chemically modified therapeutic proteins: [1a,30] the ketone of 7 can react with hydrazides or alkoxyamines,a nd the azide groups of 21 and 22 can react with alkynes such as dibenzocyclooctyne (DBCO) via copper-free click reactions. [31] Amino thiols 23, 24,and 25 can be used for site-specific labeling via oxidative coupling reactions, [12] and dithiol 31 can be selectively modified by means of at hiol-chlorine nucleophilic substitution reaction. [25a] In addition, p-nitrophenyl thiol 3 may be useful for increasing protein immunogenicity, [32] and pcyanophenyl thiol 4 could be used as an infrared probe.…”

“…[11] This system was further optimized by the Xiao group to allow functionalization of the resulting proteins by means of an oxidative conjugation reaction. [12] In 2011, Qu et al biosynthesized ad emethylated pyrrolysine analog by partially reconstituting the pyrrolysine biosynthetic pathway in E. coli and hijacking the pathway with d-ornithine.T he resulting protein can be chemically functionalized with various molecules. [13] Thes ystem was later modified to incorporate an alkyne-bearing pyrrolysine analog derived from 3S-ethynyl-d-ornithine by the Carell group.…”

Proximity‐driven, Regioselective Chemical Modification of Peptides and Proteins

“…The side chain of ncAAs could introduce chemical reactivities that are completely orthogonal to natural amino acids, providing specificity to the subsequent modification [31,32] . Some recent developments include the incorporation of a cyclopropene derivative of lysine (CypK) and its subsequent modification via an inverse‐electron‐demand Diels‐Alder reaction with a tetrazine, [33,34] and a p‐amino‐phenylalanine (pAF)‐based relay system that enables the incorporation of pAF into proteins without exogenous feeding of this amino acid [35] . The enzymatic approaches have involved peptide ligases and modification enzymes.…”