non-chromatographic-purification-recombinant-elastin-like

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from <em>Escherichia coli</em>

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Duke University

This unit presents a recombinant protein purification method that employs an elastin-like polypeptide (ELP) as a purification tag. ELPs undergo a sharp and reversible phase transition when heated above their lower critical solution temperature. ELPs retain this behavior when they are fused to a protein, and thereby provide a simple method to isolate a recombinant ELP fusion protein from cell contaminants by cycling the solution through the insoluble and soluble phase of the ELP fusion protein using a procedure that is termed Inverse Transition Cycling. This method does not require the use of chromatography, so it is cost-effective, easy to scale up, and easy to multiplex.

Elastin-like polypeptides as a purification tag for recombinant proteins.

Elastin-like polypeptides (ELPs) are a class of stimulus-responsive biopolymers whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. The lower critical solution temperature (LCST) behavior of ELPs allows them to be utilized as soluble macromolecules below their LCST, or as self-assembled nanoscale particles such as micelles, micron-scale coacervates, or viscous gels above their LCST, depending on the ELP architecture. As each ELP sequence is specified at its genetic level, functionalization of an ELP with peptides and proteins is simple to accomplish by the fusion of a gene encoding an ELP with that of the peptide or protein of interest. Protein ELP fusions, where the appended protein serves a therapeutic or targeting function, are suitable for applications in which the ELP can improve the systemic pharmacokinetics and biodistribution of the protein, or can be used as an injectable depot for sustained, local protein delivery. Here we describe considerations in the design of therapeutic protein ELP fusions and provide details of their gene design, expression, and purification.

Fusions of elastin-like polypeptides to pharmaceutical proteins.

To address the limited tumor penetration of nanoparticle drug delivery vehicles, we report the first pH-responsive polypeptide micelle that dissociates at the low extracellular pH of solid tumors. This histidine-rich elastin-like polypeptide block copolymer self-assembles at 37 °C into spherical micelles that are stabilized by Zn(2+) and are disrupted as the pH drops from 7.4 to 6.4. These pH-sensitive micelles demonstrate better in vivo penetration and distribution in tumors than a pH-insensitive control.

Triple stimulus-responsive polypeptide nanoparticles that enhance intratumoral spatial distribution.

We report herein the unexpected temperature triggered self-assembly of proteins fused to thermally responsive elastin-like polypeptides (ELPs) into spherical micelles. A set of six ELP block copolymers (ELP(BC)) differing in hydrophilic and hydrophobic block lengths were genetically fused to two single domain proteins, thioredoxin (Trx) and a fibronectin type III domain (Fn3) that binds the α(v)β(3) integrin. The self-assembly of these protein-ELP(BC) fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM. Self-assembly of the ELP(BC) was unexpectedly retained upon fusion to the two proteins, resulting in the formation of spherical micelles with a hydrodynamic radius that ranged from 24 to 37 nm, depending on the protein and ELP(BC). Cryo-TEM images confirmed the formation of spherical particles with a size that was consistent with that measured by light scattering. The bioactivity of Fn3 was retained when presented by the ELP(BC) micelles, as indicated by the enhanced uptake of the Fn3-decorated ELP(BC) micelles in comparison to the unimer by cells that overexpress the α(v)β(3) integrin. The fusion of single domain proteins to ELP(BC)s may provide a ubiquitous platform for the multivalent presentation of proteins.

Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers.

Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation.

Elastin-like polypeptides (ELPs) are thermally sensitive peptide polymers that undergo thermally triggered phase separation and this behavior is imparted to soluble proteins when they are fused to an ELP. The transition temperature of the ELP fusion protein is observed to be different than that of a free ELP, indicating that the surface properties of the fused protein modulate the thermal behavior of ELPs. Understanding this effect is important for the rational design of applications that exploit the phase transition behavior of ELP fusion proteins. We had previously developed a biophysical model that explained the effect of hydrophobic proteins on depressing the transition temperature of ELP fusion proteins relative to free ELP. Here, we extend the model to elucidate the effect of hydrophilic proteins on the thermal behavior of ELP fusion proteins. A linear correlation was found between overall residue composition of accessible protein surface weighted by a characteristic transition temperature for each residue and the difference in transition temperatures between the ELP protein fusion and the corresponding free ELP. In breaking down the contribution of residues to polar, nonpolar, and charged, the model revealed that charged residues are the most important parameter in altering the transition temperature of an ELP fusion relative to the free ELP.

Predicting transition temperatures of elastin-like polypeptide fusion proteins.

Calcium-sensitive elastin-like polypeptides (CELPs) were synthesized by periodically interspersing a calcium-binding peptide sequence from calmodulin within an elastin-like polypeptide (ELP) with the goal of creating thermal and calcium responsive peptide polymers. The CELPs exhibit high sensitivity to calcium compared to monovalent cations but do not exhibit the exquisite selectivity for calcium over other divalent cations, such as magnesium, that is displayed by calmodulin. The CELPs were further used as a building block for the synthesis of calcium-sensitive nanoparticles by fusing a hydrophilic, noncalcium-sensitive ELP block with a CELP block that becomes more hydrophobic upon calcium binding. We show that addition of calcium at concentrations between 50 and 500 mM imparts sufficient amphiphilicity to the diblock polypeptide between 33 and 46 °C to trigger its self-assembly into monodisperse spherical micelles with a hydrodynamic radius of ∼50 nm.

Calcium binding peptide motifs from calmodulin confer divalent ion selectivity to elastin-like polypeptides.

It is shown that hydrophilic (backbone) and hydrophobic (side chain) hydration layers of elastin-like polypeptides (ELPs), a class of stimulus responsive peptide polymers that exhibit lower critical solution temperature (LCST) phase transition behavior, can exist in a coupled and decoupled state. The decoupled hydration state consists of hydrophobic and hydrophilic hydration layers that respond independently to temperature, while the coupled hydration state is characterized by a common, cooperative dehydration of both hydration layers. It is further shown that the primary sequence of an ELP can be tuned to exhibit either of the hydration layer coupling modes. Charged side chains lead to decoupling, while strongly hydrophobic side chains trigger stronger interaction between hydrophilic and hydrophobic hydration, leading to coupling of both layers. Further, for aprotic residues this coupling is fostered by decreasing bulkiness of hydrophobic side chains due to larger hydration numbers and water molecules mediating coupling between side chain and backbone hydration shells. For coupled hydration shells, the LCST phase transition characterized by spin probing continuous wave electron paramagnetic resonance spectroscopy is reminiscent of a first-order process even on nanoscopic length scales. In contrast, analogous synthetic polymers exhibit nanoscale phase transitions over a broad temperature range, indicating that their nanoscale phase behavior is not of first order. Hence, our results indicate that ELPs are the first identified class of polymers that exhibit a first-order inverse phase transition on nanoscopic length scales. These results may also provide insights into the role of hydration layers in governing the structure-function relationship of intrinsically disordered proteins.

Wafa Hassouneh

Department of Biomedical Engineering,
Research Triangle MRSEC

Duke University

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Wafa Hassouneh

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering,Research Triangle MRSEC

Duke University

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Department of Biomedical Engineering,
Research Triangle MRSEC