Diverse protein manipulations with genetically encoded glutamic acid benzyl ester

Abstract

Site-specific modification of proteins has significantly advanced the use of protein in biological research and therapeutics development. Among various strategies aimed at this end, genetic code expansion (GCE) allows structurally...

Site‐Specific Protein Modification with Reducing Carbohydrates

“…The naturally evolved pyrrolysyl‐tRNA synthetase/tRNA pair (PylRS/PylT) could incorporate pyrrolysine into proteins in response to amber codon [23] and has been extensively engineered to encode a large number of ncAAs in different organisms [24] . To encode BnD, a previously constructed Methanosarcina barkeri PylRS library (residues A267, Y271, N311, C313 and Y349 randomized to NNK, N=A, T, G or C, K=T or G) was subjected to multiple rounds of positive and negative selection in E. coli DH10B as has been described [22, 25] . To our delight, colonies that showed BnD‐dependent survival were obtained from the last round of selection, and DNA sequencing revealed 6 distinct PylRS variants (BnDRS1‐6, Table S1).…”

“…Curiously, we wondered whether this Endo M variant could accept other hydrazide‐linked GlcNAcs on proteins. To this end, we incorporated glutamic acid benzyl ester (BnE) at Q62 in Ub‐K63A and converted it into the corresponding hydrazide (L‐α‐aminobutyric acid γ hydrazide, Bγz) [25] . Bγz on Ub could react with GlcNAc in a similar manner to Aβz with the formation of Ub‐Q62Bγz‐GlcNAc‐K63A (Figure S10).…”

“…[24] To encode BnD, a previously constructed Methanosarcina barkeri PylRS library (residues A267, Y271, N311, C313 and Y349 randomized to NNK, N = A, T, G or C, K = T or G) was subjected to multiple rounds of positive and negative selection in E. coli DH10B as has been described. [22,25] To our delight, colonies that showed BnD-dependent survival were obtained from the last round of selection, and DNA sequencing revealed 6 distinct PylRS variants (BnDRS1-6, Table S1). Furthermore, in a GFP-based fluorescence assay, all these variants could enhance the fluorescence intensity in the presence of BnD (Figure S1).…”

“…To this end, we incorporated glutamic acid benzyl ester (BnE) at Q62 in Ub-K63A and converted it into the corresponding hydrazide (L-α-aminobutyric acid γ hydrazide, Bγz). [25] Bγz on Ub could react with GlcNAc in a similar manner to Aβz with the formation of Ub-Q62Bγz-GlcNAc-K63A (Figure S10). The following transglycosylation reaction revealed that Bγz-GlcNAc on Ub could accept SCT-ox based on HPLC-MS analysis with relatively good efficiency (45 %, Figure S11).…”

A Shelf Stable Fmoc Hydrazine Resin for the Synthesis of Peptide Hydrazides

“…17 This method avoids the need for strong oxidizing conditions, enabling broader compatibility with common synthetic peptide functionalities, and has been widely adopted in a broad range of applications. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] The increased use of C-terminal α-hydrazides in peptide chemistry creates a demand for robust synthetic tools for accessing these moieties. A number of methods have been published to allow access to C-terminal hydrazides on both synthetic 15,16,38 and expressed 15,39,40 peptides and proteins.…”