Site-Specific Modification of Recombinant Proteins: A Novel Platform for Modifying Glycoproteins Expressed in E. coli

Abstract

The site-specific modification of proteins is expected to be an important capability for the synthesis of bioconjugates in the future. However, the traditional repertoire of reactions available for the direct modification of proteins suffers from lack of specificity, necessitating costly downstream processing to isolate the specific species of interest. (1) Here, we use a well-established, glycan-specific chemistry to PEGylate model glycoproteins, each containing a unique reactive GalNAc attached to a specifically engineered threonine residue. By engineering E. coli to execute the initial steps of human, mucin-type O-glycosylation, we were able to obtain homogeneous site-specifically modified glycoproteins with fully human glycan linkages. Two mucin-based reporters as well as several fusion proteins containing eight-amino-acid GalNAc-T recognition sequences were glycosylated in this engineered glycocompetent strain of E. coli. The use of one sequence in particular, PPPTSGPT, resulted in site-specific glycan occupancy of approximately 69% at the engineered threonine. The GalNAc present on the purified glycoprotein was oxidized by galactose oxidase and then coupled to hydroxylamine functionalized 20 kDa PEG in the presence of aniline. The glycoprotein could be converted to the PEGylated product at approximately 85% yield and >98% purity as determined by comparison to the products of control reactions.

Pegylation and Its Impact on the Design of New Protein-Based Medicines

“…In addition to cysteine-specific conjugation, other amino acids (e.g., arginine, tyrosine and glutamic acid) [119][120][121][122] and glycosyl moieties [123][124][125][126][127][128] have also been described as targets for conjugation. PEGylation of either the C- [129] or the N-terminus [86,88,90,[130][131][132][133][134] of proteins has also been described.…”

Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

“…5,[7][8][9] GalOx is currently used in several biotechnological applications, due to its selectivity and unique mode of action of producing a reactive aldehyde functionality and hydrogen peroxide. It has been employed, for example, as a possible dental anti-plaque system 10,11 and in various analytical methods for the determination of lactose and other galactosides, [12][13][14] in glycoprotein detection and bioconjugation, 15,16 and in disease diagnostics. 17,18 Likewise, GalOx has been used for chemo-enzymatic polysaccharide functionalization in the production of novel biopolymers and cellulosic materials.…”

Chemobacterial Synthesis of a Sialyl‐Tn Cyclopeptide Vaccine Candidate

“…In vertebrates, STn biosynthesis begins with the enzymatic addition of α‐GalNAc from UDP‐GalNAc, involving a specific family of α‐GalNAc transferases (GalNAc‐T), to hydroxylated amino‐acid Thr/Ser residues in a mucin‐specific domain. A truncated derivative of human GalNAc‐T2 was recently expressed in E. coli to perform in vivo mucin O‐glycosylation of specific peptides . However, this particular approach was not appropriate here, because we sought to synthesise the smallest STn epitope suitable for the design of a multivalent cyclopeptide platform without the large mucin sequence required for GalNAc‐T activity.…”