Browsing by Subject "Elastin-like polypeptides"

Over 30 million people in the United States suffer from type 2 diabetes (T2D), and this figure is rapidly increasing. Currently available glucose-lowering drugs largely treat the symptoms of diabetes and not the underlying pathology, leaving one third of diabetes patients with improperly managed disease. Thus, there exists an urgent need for novel drugs that slow T2D progression while posing a minimal burden on the patient.

The metabolic regulatory factor fibroblast growth factor 21 (FGF21) is under investigation as a T2D therapeutic due to its favorable effects on glycemic control and body weight. However, the feasibility of native FGF21 as a drug candidate is impeded by its rapid in vivo clearance and by costly production methods associated with poor protein solubility. To address these issues, FGF21 was recombinantly expressed in E. coli as a fusion with an elastin-like polypeptide (ELP), a repetitive peptide polymer with reversible thermal phase behavior. Below their transition temperature (Tt), ELPs exist as soluble unimers, while above their Tt, they aggregate into an insoluble coacervate. The thermal responsiveness of the ELP was retained when genetically fused to FGF21, with several notably positive impacts for the synthesis and efficacy of this protein drug. First, the ELP fusion partner acted as a solubility enhancer, yielding 50 mg/L of active FGF21 protein from the soluble cell lysate fraction in shaker flask culture, and eliminating the need for protein refolding. Second, the phase transition behavior of the ELP was exploited for chromatography-free FGF21 purification. Third, the Tt of the ELP was tuned to below body temperature, such that the phase transition was initiated solely by body heat. Indeed, in vivo injection of the fusion resulted in an immiscible viscous phase in the subcutaneous (s.c.) space that dissolved at a steady rate, temporally releasing fusion unimers into circulation. The injectable FGF21 drug depot was tested in diabetic ob/ob mice, and conferred dose-dependent glucose-and weight-lowering effects that were sustained out to 5 days following a single s.c. injection.

Once an optimized ELP-based FGF21 delivery strategy was established, the fusion concept was applied to a combination therapy to afford even greater metabolic benefits, while providing controlled release properties exclusive to the ELP platform. Recent evidence supports the development of combination drug treatments that incorporate complementary mechanisms of action to more effectively treat T2D. Thus, we developed a unimolecular dual agonist by combining the incretin glucagon-like peptide-1 (GLP1) with FGF21, hypothesizing that this agent would merge the insulinotropic and anorectic effects of GLP1 with the enhanced insulin sensitivity and energy expenditure associated with FGF21 signaling. The dual agonist was designed as a single polypeptide fusion, with GLP1 located at the N terminus and FGF21 at the C terminus. This orientation allowed each peptide to activate its endogenous receptor, while the linear architecture enabled facile synthesis in a bacterial expression system. An ELP was fused between GLP1 and FGF21 to serve as both a flexible linker and a depot-forming delivery scaffold. Indeed, a single s.c. injection of GLP1-ELP-FGF21 into diabetic db/db mice resulted in potent metabolic effects that were sustained at least 7 days, indicating formation of an ELP depot with a highly controlled rate of drug release. Furthermore, dual agonist treatment outperformed a long-acting GLP1 analog in restoring glycemic control and inducing weight loss, supporting the rationale for a GLP1/FGF21 combination therapy.

With a significant proportion of T2D patients failing to properly manage their disease, there is an urgent need for novel drug and drug combinations that effectively target disease pathophysiology, while posing a minimal burden on the patient. Meanwhile, the vast – and global – prevalence of metabolic disease argues for cost-effective and scalable manufacturing methods for new drugs. An ELP-based approach to therapeutics precisely addresses these needs by providing a streamlined method for production, as well as an innovative strategy for drug delivery to reduce the frequency of administration and thereby promote patient compliance. Furthermore, the ELP platform can be utilized to unite distinct drugs into one multi-functioning molecule to more effectively treat diabetes, altogether simplifying and improving metabolic disease management.

Surfaces with switchable properties in response to external stimuli (e.g., temperature and pH) have attracted substantial research interest because of their ability to modulate biomolecule activity, protein immobilization, and cell adhesion. These stimuli-responsive substrates offer versatile platforms for developing biosensors, cell culture substrates, diagnostic systems, and drug delivery systems. In this work, we controllably functionalized substrates with genetically engineered polypeptides to fabricate thermally responsive surfaces for various bioanalytical applications. Genetically engineered elastin-like polypeptides (ELPs) are one class of thermally responsive biopolymers that are characterized by their lower critical solution temperature (LCST) phase behavior in water; ELPs at a given concentration in aqueous solvent phase separate to form protein-rich coacervates above the cloud point transition temperature (Tt). ELPs present an attractive alternative to synthetic, stimuli-responsive polymers due to their biocompatibility, monodispersity, and controlled physicochemical properties.

To fabricate ELP-modified surfaces with desired structure and functionality, we first investigated the adsorption behavior of ELP homopolymers and ELP block copolymers onto silica surfaces. We provided an in-depth understanding of adsorption kinetics, mechanism and surface conformation for the “canonical” ELP sequence (Val-Pro-Gly-Val-Gly), which enabled precise conformational control of the adsorbed ELPs. We also showed that genetically incorporating the silaffin R5 peptides into ELP chains significantly enhanced the binding affinity of ELPs to silica surfaces, leading to thicker ELP layers with a higher surface coverage. To extend this work, we also explored the adsorption behavior of ELP block copolymers onto silica surfaces using theoretical and experimental approaches. Our results showed that the silaffin tag not only enhanced the binding of ELP block copolymers to silica surfaces, but also directed micelle adsorption, leading to close-packed micellar arrangements dissimilar to the sparse and patchy arrangements observed for ELP micelles lacking a silaffin tag. In addition, the surface-grafted ELP unimers exhibited interfacial phase transition behavior, while the adsorbed ELP micelles were no longer thermally-responsive. These studies provided insight into the design of ELP based smart surfaces with controlled structure-architecture-function relationship.

After achieving programmable adsorption of ELPs onto surfaces, we exploited these thermally responsive surfaces for several bioanalytical applications, including cell culture and diagnostic assays. We first developed a simple approach to pattern cells on gold patterned silicon substrates using ELPs with cell- and gold-binding domains. Cell patterning was achieved by exploiting orientation of the adsorbed ELP to either enhance (gold regions) or impede (silicon oxide regions) cell adhesion at particular locations on the patterned surface. Along a similar vein, we fabricated a thermoresponsive cell culture substrate using rationally designed ELP coatings with precisely spaced cell-adhesive motifs. The reversible swelling and collapse of ELPs thermally modulated the accessibility of cell-binding domains to enable cell adhesion at T > Tt and efficient cell recovery at T < Tt.

In addition, we have utilized ELP-modified particles to develop smart diagnostics. We demonstrated proof-of-concept for an acoustofluidic, chip-based method that enables the rapid capture and isolation of biomarkers from blood for off-chip quantification. We showed that biomarkers were rapidly immobilized onto the surfaces of ELP-modified particles via co-aggregation, and continuously separated from the blood cells using an acoustofluidic device. The captured biomarkers can then be quantified using flow cytometry, or released from the surfaces of particles for further analysis. By designing ELP fusion proteins that can capture target bioactive materials, this platform system can be readily extended to separate a range of biological materials (e.g., cells, viruses and cell-free DNA) from complex biofluids.

In summary, we achieved controlled adsorption of ELP homopolymers and block copolymers onto surfaces with tailored architecture and functionality. These ELP-modified smart surfaces have been utilized to create cellular patterns, a thermoresponsive cell culture substrate, and a biomarker separation and detection platform.

Disordered proteins lack a defined secondary and higher order structure, possess a large number of biological functions, and are prevalent throughout nature. One important property of certain disordered proteins in biology is an ability to phase separate within the interior of a cell and form liquid non-membrane bound organelles. These compartments are thought to play crucial roles in various cellular processes such as ribonucleic acid maintenance and gene expression. While the physicochemical drivers and biophysical properties of intracellular organelles is becoming clear, their functions remain poorly understood due to the difficulty in probing them and the lack of engineered models of them.

This work addresses the need for engineered systems of membraneless organelles and consists of programming the assembly of elastin-like polypeptides, an archetypal class of disordered proteins, into model organelles within droplet microenvironments. We synthesized a library of recombinant elastin-like polypeptides, and studied their phase separation and assembly into various organelles, from layered to mixed to size-controlled, within water microdroplets using lightfield, darkfield, confocal, and fluorescence microscopy. We coupled this with bulk characterization techniques, in the form of spectrophotometry and light scattering, to develop a theoretical framework for understanding and predicting disordered protein phase separation into these biologically relevant structures. Furthermore, we adapted our findings to engineer new thermoresponsive colloidal gels with size tunable across four orders of magnitude (nano-to-meso-to-microscale) that are comprised of elastin-like polypeptides containing unnatural amino acid photocrosslinkers. Lastly, we developed model functional ribonucleoprotein organelles composed of RNA-binding elastin-like polypeptides designed de novo, and show that these organelles temporally regulate gene expression within droplet-based protocells. Taken together, this body of work offers new insights into (1) our understanding of the genetic to molecular to macroscale relationships encoding naturally occurring intrinsically disordered protein assemblies, (2) the design rules of these assemblies in cell biology, and it enables (3) the facile engineering of unprecedented polypeptide biomaterials in the form of (i) thermoresponsive photocrosslinked colloidal gels with potential utility in drug delivery and (ii) functional, artificial ribonucleoprotein granules that lay the foundation for developing more complex, realistic artificial cells.

Despite decades of research since the discovery of the environmental sensitivity of tropoelastin, only a handful of elastin-inspired polypeptides departing from the canonical VPGXG motif, where X is any amino acid except proline, have been uncovered. Hence, the field of "smart" protein-polymers has evolved mainly through the introduction of innovative molecular architectures. Instead, we decided to explore sequence diversity as a necessary tool to broaden the biomedical and biotechnological utility of these "smart" protein-polymers. Using a new, highly parallel method for the synthesis of repetitive genes, we conducted a systematic study of the sequence constraints of the canonical VPGXG motif by substituting or inserting Alanine residues along this pentapeptide motif, which yielded new pentapeptide and hexapeptide, non-canonical ELP motifs. These studies led to the discovery of new families of hexapeptide motifs with fully reversible phase transition behavior and suggested an unexpected degree of sequence and conformational promiscuity in the canonical motif that hints at the existence a large space of amino acid sequences with intrinsic "smart" behavior. Moreover, this work shed light into the conformational requirements of the phase transition behavior and suggested the possibility to control the assembly of "smart" protein-polymers in a sequence-controlled manner.

Elastin-like polypeptides (ELPs) are thermally responsive polymers composed of the pentapeptide repeat Valine-Proline-Glycine-X-Glycine where X is any amino acid except proline. ELP diblocks have been engineered by creating two ELP blocks with hydrophilic and hydrophobic guest residues. The hydrophobic block desolvates at a lower temperature and forms the core of a micelle while the still hydrated hydrophilic block forms the corona. ELP micelles are promising drug delivery vehicles for cancer therapeutics. ELP diblocks offer a unique method to display targeting proteins multivalently on micelles to improve tumor cell uptake. As ELPs are genetically encoded, proteins can be seamlessly fused at the genetic level to the ELP diblock. The protein ELP diblock fusions can be synthesized as one polypeptide chain that is of precise molecular weight and highly monodisperse, and no post-synthesis modification is necessary. Self-assembly behavior of ELP diblocks is known to tolerate fusion to small peptides (< 10 amino acids) but their self-assembly behavior has not be examined when fused to proteins that are 100-200 amino acids. Here, we hypothesize that molecular weight of the protein and the surface properties of the protein will be factors in determining its effect on ELP diblock self-assembly. In addition, the ELP block lengths and composition are hypothesized to be factors in the self-assembly behavior of protein ELP diblock fusions. This hypothesis is tested by fusing four proteins with different properties to various ELP diblocks and characterizing their self-assembly behavior. The proteins were found to dominate the self-assembly behavior. Proteins that disrupted self-assembly did so for all ELP diblock lengths and compositions. Protein that did not disrupt self-assembly behavior affected the thermal behavior of the hydrophilic block. Hydrophilic proteins increased the micelle-to-aggregate transition temperature while hydrophobic proteins decreased it. We also sought to understand the self-assembly of ELP diblocks on a theoretical basis. A previously developed model for the self-assembly of synthetic polymers was applied to our polypeptide system. Two parameters, solvent quality of the corona and surface tension of the hydrophobic block, were experimentally measured and used to fit the model. Predictions of micelle radius and aggregation numbers were in good agreement with experimental data. However, the corona was found to be unstretched compared to its Gaussian size by this model. Therefore, a new model was developed describing what is termed as weak micelles in which the corona is not stretched but rather close to Gaussian size. The weak micelle model prediction were also in good agreement with experimental data suggesting that ELP micelles are in the crossover regime between the previous model and the new model.