Sortase‐Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein

Abstract

Conventional methods for synthesizing protein/peptide–polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site-specificity, which results in heterogeneous products with significantly compromised bio activity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with > 90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide–polymer conjugates.

Protein–polymer conjugation — moving beyond PEGylation

“…Chilkoti and coworkers were the first to demonstrate grafting of a single chain of POEGMA from a defined site–the N or C terminus– of a protein using ATRP under aqueous conditions to yield site-specific and stoichiometric (1:1) conjugates with relatively low polydispersity and high yield [39–41●●]. These protein-POEGMA conjugates showed a 15–50 fold increase in blood exposure [39●●] and 50 fold increase in tumor accumulation [40●●] compared to the unmodified proteins upon intravenous administration to mice, thereby demonstrating that comb polymers that present short oligo(ethylene glycol) side-chains are a new class of PEG-like polymers that can significantly improve the pharmacological properties of proteins.…”

Site-selective in situ growth of fluorescent polymer–antibody conjugates with enhanced antigen detection by signal amplification

“…ATRP reactions were performed using conditions described previously with minor changes [25,26,30]. OEGMA (MW ¼ 500) was purified by running through a column packed with aluminum oxide to remove the inhibitor.…”

Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

“…Notable examples include the ligation of proteins or peptides to fluorophores (Popp et al, 2007; Yamamoto & Nagamune, 2009), carbohydrates (Samantaray, Marathe, Dasgupta, Nandicoori, & Roy, 2008; Wu, Guo, Wang, Swarts, & Guo, 2010), polymers (Parthasarathy et al, 2007; Qi, Amiram, Gao, McCafferty, & Chilkoti, 2013), solid supports (Chan et al, 2007; Le, Raeeszadeh-Sarmazdeh, Boder, & Frymier, 2015; Sinisi et al, 2012), therapeutics (Beerli, Hell, Merkel, & Grawunder, 2015; Fang et al, 2016), lipids (Antos et al, 2008; Wu et al, 2010), nucleic acids (Koussa, Sotomayor, & Wong, 2014; Pritz et al, 2007), metal chelators (Paterson et al, 2014; Westerlund, Honarvar, Tolmachev, & Eriksson Karlström, 2015), viral particles (Hess et al, 2013; Schoonen et al, 2015), live cells (Popp et al, 2007; Shi et al, 2014; Yamamoto & Nagamune, 2009), or even intramolecular ligations for generating cyclic proteins and peptides (Antos et al, 2009b; Jia et al, 2014; van ’t Hof et al, 2015). A full discussion of all relevant applications is beyond the scope of this unit, and we refer to the reader to reviews on this topic (Haridas, Sadanandan, & Dheepthi, 2014; Popp & Ploegh, 2011; Ritzefeld, 2014; Schmohl & Schwarzer, 2014).…”