Protein PEGylation: Navigating Recombinant Protein Stability, Aggregation, and Bioactivity

Abstract

Enzymes play a powerful role as catalysts with high specificity and activity under mild environmental conditions. Significant hurdles, such as reduced solubility, reduced shelf-life, aggregate formation, and toxicity, are still ongoing struggles that scientists come across when purifying recombinant proteins. Over the past three decades, PEGylation techniques have been utilized to significantly overcome low solubility; increased protein stability, shelf-life, and bioactivity; and prevented protein aggregate formation. This review seeks to highlight the impact of PEG-based formulations that are significantly utilized to obtain favourable protein physiochemical properties. The authors further discuss other techniques that can be employed such as coexpression studies and nanotechnology-based skills to obtaining favourable protein physiochemical properties.

Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates

“…( 51 ), increases the net size, hydrodynamic radius, and the molecular weight of proteins and also alters their physicochemical properties like spatial hindrance, conformation, and electrostatic binding. Successful pegylation results in decreased systemic clearance, macrophage uptake, and proteolysis of the proteins, as well as decreased immunogenicity ( 52 ). For example, pegylation of the hGH antagonist B2036 with 4 to 6 5-kDa PEG moieties on random lysines and the N -terminal phenylalanine (pegvisomant) significantly decreased B2036’s binding affinity by about 20-fold but greatly increased its circulating half-life in humans to ∼70-h from 20-min ( 14 ).…”

Tailored Branched Polymer–Protein Bioconjugates for Tunable Sieving Performance

“…The traditional “grafting-to” strategy has long been used to attach presynthesized polymers like poly­(ethylene glycol) (PEG) to proteins, reducing immunogenicity by masking antibody binding sites. , However, PEGylation strongly inhibits the cellular absorption and escape from endosomes, leading to a reduction in the effectiveness of the delivery system. ,, In contrast, the “grafting-from” approach involves growing polymers directly from the protein surface. Atom transfer radical polymerization (ATRP) has been widely used for grafting-from proteins, enabling high grafting density, site-specific polymer growth, and the rational synthesis of protein–polymer conjugates with improved solubility, stability, and functionality. − …”

Enhancing Protein Solubility via Glycosylation: From Chemical Synthesis to Machine Learning Predictions

“…These strategies encompass the utilization of additives in buffers, such as ionic compounds, 16 salts, 17 and detergents, 18 as well as the mutation of hydrophobic residues to hydrophilic counterparts 19 attachment of polyethylene glycol (PEG) to protein side chains. 20 In addition to these strategies, naturally occurring post-translational modifications (PTMs) of proteins have shown promise in improving protein solubility. 21 Among these PTMs, glycosylation, an important type of modification, has been found to significantly enhance protein solubility.…”

“…Several strategies have been developed and investigated in previous studies to enhance protein solubility. These strategies encompass the utilization of additives in buffers, such as ionic compounds, salts, and detergents, as well as the mutation of hydrophobic residues to hydrophilic counterparts and the attachment of polyethylene glycol (PEG) to protein side chains . In addition to these strategies, naturally occurring post-translational modifications (PTMs) of proteins have shown promise in improving protein solubility .…”