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.

Advances in the Generation of Constructed Cardiac Tissue Derived from Induced Pluripotent Stem Cells for Disease Modeling and Therapeutic Discovery

“…iPSC-CCTs and cardiac tissue stimulation produce exosomes tailored to specific tissue healing [3,33,35,[91][92][93]99]. Similar to ECM-targeting peptides used for wound healing, like PLGF-2 derivatives [98,102], cardiac homing peptides (CHPs) specifically target ischemic myocardial tissue in ExT, improving MI recovery [33,35,78,92,93,103]. Non-invasive delivery with catheters, intravenous injections, and nebulizers facilitates the clinical translation of ExT [93,99].…”

Advances in Therapeutic Peptides Separation and Purification

“…Truman and colleagues reported enhanced yields for separating elastin-like proteins using pI-based phase separation (pI-BPS) [106]. This method leverages the proteins' pI, exposing them to conditions where their net charge is zero.…”

“…This method leverages the proteins' pI, exposing them to conditions where their net charge is zero. This induces aggregation or phase separation of the proteins from the solution, facilitating their purification [106].…”

Genetically Fusing Order‐Promoting and Thermoresponsive Building Blocks to Design Hybrid Biomaterials

“…[19,20] ELPs have been widely used as a purification tag in inverse transition cycling (ITC) purification due to their reversible phase transition behavior and provide a simple, nonchromatographic method for the purification of proteins. [21,22] As a extracellular matrix (ECM) protein-derived material, ELPs show low cytotoxicity and great biocompatibility. [7,23] Hence they have been considered a promising building block for drug carriers.…”