Transforming protein-polymer conjugate purification by tuning protein solubility

Abstract

Almost all commercial proteins are purified using ammonium sulfate precipitation. Protein-polymer conjugates are synthesized from pure starting materials, and the struggle to separate conjugates from polymer, native protein, and from isomers has vexed scientists for decades. We have discovered that covalent polymer attachment has a transformational effect on protein solubility in salt solutions. Here, protein-polymer conjugates with a variety of polymers, grafting densities, and polymer lengths are generated using atom transfer radical polymerization. Charged polymers increase conjugate solubility in ammonium sulfate and completely prevent precipitation even at 100% saturation. Atomistic molecular dynamic simulations show the impact is driven by an anti-polyelectrolyte effect from zwitterionic polymers. Uncharged polymers exhibit polymer length-dependent decreased solubility. The differences in salting-out are then used to simply purify mixtures of conjugates and native proteins into single species. Increasing protein solubility in salt solutions through polymer conjugation could lead to many new applications of protein-polymer conjugates.

A novel L‐asparaginase from the symbiotic Enterobacter aerogenes isolated from Eucheuma sp.

“…The protein content of the crude extract was 0.298 mg/ml (Table 5). quality of ammonium sulfate added (Baker et al, 2019;Green & Hughes, 1955;Sha et al, 2014). The highest concentration of protein was found at a 0%-20% fraction of 5.17 mg/ml.…”

“…Table 5 shows that each fraction had different protein concentrations, which means that protein precipitated from each fraction had different proteins. The protein is precipitated based on differences in water solubility, higher solubility in water, and quality of ammonium sulfate added (Baker et al, 2019; Green & Hughes, 1955; Sha et al., 2014). The highest concentration of protein was found at a 0%–20% fraction of 5.17 mg/ml.…”

Nanohybrids as Protein-Polymer Conjugate Multimodal Therapeutics

“…Since proteins are least soluble at their isoelectric point, any modifications to protein structure can alter the solubility of the protein. Different intrinsic and extrinsic factors influence the solubility of the protein corresponding to the number of charged amino acids and the chemical structure on the protein surface (51). A few amino acids like lysine, tyrosine, disulfide-bonded cysteine as well as the modified C-and N-terminal amines have importance in residue-specific and site-selective conjugation (52)(53)(54)(55).…”

Aglycone sterics-selective enzymatic glycan remodeling

“…With these criteria in mind, we herein devise a multi-faceted modulation platform for glycoenzyme accessibility, by means of polymer-based, controlled single-enzyme caging, to achieve and regulate the aglycone-steric selectivity of glycan remodeling. Owing to the feature of tailored synthesis and well-defined properties of synthetic polymers ( Liu and Gao, 2021 ; Lu et al., 2020 ; Kaupbayeva and Russell, 2020 ), wrapping up protein with a polymer shell has been extensively used to alter protein’s bioactivity and stability ( Heredia et al., 2005 ; Lele et al., 2005 ; Cummings et al., 2013 ; Murata et al., 2013 ; Kovaliov et al., 2018 ), tune solubility ( Baker et al., 2019 ), improve circulating half-life ( Harris and Chess, 2003 ), decrease immunogenicity ( Liu et al., 2014 ), and adjust substrate/inhibitor affinity ( Liu et al., 2013 ; Murata et al., 2014 ; Kaupbayeva et al., 2019 ). More importantly, different regulation mechanisms can be introduced to proteins by conjugation with stimuli-responsive polymers ( Heredia et al., 2005 ; Cummings et al., 2013 ; Murata et al., 2013 ; Chen and Hoffman, 1993 ; Stayton et al., 1995 ; Shimoboji et al., 2002 , 2003 ; Boyer et al., 2007 ; De et al., 2008 ; Mackenzie and Francis, 2013 ; Gobbo et al., 2018 ).…”