Isoelectric point (pI)-based phase separation (pI-BPS) purification of elastin-like polypeptides (ELPs) containing charged, biologically active fusion proteins (ELP-FPs).

Abstract

Elastin-like polypeptides (ELPs) are peptide-based biomaterials with residue sequence (VPGXG)n where X is any residue except proline. ELPs are a useful modality for delivering biologically active proteins (growth factors, protease inhibitors, anti-inflammatory peptides, etc.) as fusion proteins (ELP-FP). ELP-FPs are particularly cost-effective because they can be rapidly purified using Inverse Temperature Cycling (ITC) via the reversible formation and precipitation of entropically driven aggregates above a transition temperature (Tt ). When ELP fusion proteins (ELP-FPs) contain significant charge density at physiological pH, electrostatic repulsion between them severely inhibits aggregate formation. The literature does not currently describe methods for purifying ELP-FPs containing charged proteins on either side of the ELP sequence as fusion partners without organic solvents. Here, the isoelectric point (pI) of ELP-FPs is discussed as a means of neutralizing surface charges on ELP-FPs and increasing ITC yield to dramatically high levels. We use pI-based phase separation (pI-BPS) to purify ELP-FPs containing cationic and anionic fusion proteins. We report a dramatic increase in protein yield when using pI-BPS for purification of ELP-FPs. Proteins purified by this method also retain the functional activity of the protein present in the ELP-FP. Techniques developed here enable significant diversification of possible fusion proteins delivered by ELPs as ELP-FPs by allowing them to be produced and purified at higher quantities and yields.