Synthesis of Polymerizable Protein Monomers for Protein-Acrylamide Hydrogel Formation

Abstract

A novel method to produce protein polymer conjugates for protein-acrylamide hydrogel formation is described. Alkenes are incorporated onto the N-terminus of expressed proteins to produce polymerizable protein monomers that can be utilized in protein-acrylamide copolymerization. A 4-vinylbenzoic acid thioester was synthesized and attached to the N-termini of two protein models, the immunoglobulin-binding protein Protein G and the bacterial enzyme xanthine-guanine phosphoribosyltransferase (GPRT), utilizing native chemical ligation. N-terminal cysteine containing proteins utilized in native chemical ligation reactions were generated from His-tagged fusion proteins using tobacco etch virus NIa (TEV) protease cleavage. The 4-vinylbenzyl functionalized proteins were good substrates for immobilizing proteins into polyacrylamide hydrogels via free radical induced protein-acrylamide copolymerization. The protein copolymerization procedures developed in this report are mild enough to allow proteins to retain measurable biological activity as demonstrated by the retention of immunoglobulin binding ability by immobilized Protein G and enzymatic activity of immobilized GPRT.

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

“…52–55 Endoglycosidase H and restriction enzymes were obtained from New England Biolabs (Ipswich, MA). Protein G resin was produced by coupling protein G (recombinantly expressed in E. coli) 56 with Sepharose ® CL-4B (Sigma-Aldrich, St Louis, MO) using divinyl sulfone as a coupling reagent. 57 General chemicals were purchased from Sigma-Aldrich and Fisher Scientific unless otherwise noted.…”

Conjugation of Methacrylamide Groups to a Model Protein via a Reducible Linker for Immobilization and Subsequent Triggered Release from Hydrogels

“…The experimental set‐up is based on the method described by Xiao and Tolbert 20. In detail, 75 µL of modified ( LMA3 ) or non‐modified LZM solution (4.25 mg · mL −1 ) was mixed with 100 µL of a 30 wt.‐% acrylamide/bisacrylamide solution (29:1 w/w), 75 µL sample buffer [0.06 M Tris‐HCl, 25 vol.‐% glycerol, 4 vol.‐% sodium dodecylsulfate (SDS), 0.1 wt.‐% bromophenol, pH = 6.8] and 50 µL PBS (pH = 5).…”

“…One approach for the covalent immobilization of proteins is the synthesis of protein macromers, which can be copolymerized in hydrogel networks. This technique is used for enzyme immobilization,18–20 as well for drug delivery purposes. For the latter application, protein‐polymer conjugates were polymerized to form hydrogels which can be used for the controlled delivery of entrapped drug molecules 21, 22.…”

Modification of polypeptide materials by Thiol-X chemistry

“…If we are thinking about peptides or polypeptides and thiol-ene chemistry, the most natural monomer to start with is cysteine. Cysteine has been used to anchor peptides and proteins to sugars [18], monomers [19], polymers [20e23], hydrogels [24,25] and nanoparticles [26]. The most impressive recent example is the 3D cell microenvironment developed by the Anseth group [24,25] Hydrogels were first formed by crosslinking a tetra-azidefunctionalized polyethylene glycol (PEG) with an enzymatically degradable polypeptide sequence that bared both an activated alkyne (di-fluorinated cyclooctyne, proving both the ring strain and electron withdrawing moieties) for copper-less click chemistry and alkene (Fmoc-Lys(alloc)-OH, protected allyl functionalized lysine) for later thiol-ene chemistry.…”