Elastin-like Proteins Research Articles

Cohesive Living Bacterial Films with Tunable Mechanical Properties from Cell Surface Protein Display.

Engineered living materials (ELMs) constitute a novel class of functional materials that contain living organisms. The mechanical properties of many such systems are dominated by the polymeric matrices used to encapsulate the cellular components of the material, making it hard to tune the mechanical behavior through genetic manipulation. To address this issue, we have developed living materials in which mechanical properties are controlled by the cell-surface display of engineered proteins. Here, we show that engineered Esherichia coli cells outfitted with surface-displayed elastin-like proteins (ELPs, designated E6) grow into soft, cohesive bacterial films with biaxial moduli around 14 kPa. When subjected to bulge-testing, such films yielded at strains of approximately 10%. Introduction of a single cysteine residue near the exposed N-terminus of the ELP (to afford a protein designated CE6) increases the film modulus 3-fold to 44 kPa and eliminates the yielding behavior. When subjected to oscillatory stress, films prepared from E. coli strains bearing CE6 exhibit modest hysteresis and full strain recovery; in E6 films much more significant hysteresis and substantial plastic deformation are observed. CE6 films heal autonomously after damage, with the biaxial modulus fully restored after a few hours. This work establishes an approach to living materials with genetically programmable mechanical properties and a capacity for self-healing. Such materials may find application in biomanufacturing, biosensing, and bioremediation.

Management of experimental workflows in robotic cultivation platforms

In the last decades, robotic cultivation facilities combined with automated execution of workflows have drastically increased the speed of research in biotechnology. In this work, we present the design and deployment of a digital infrastructure for robotic cultivation platforms. We implement a workflow management system, using Directed Acyclic Graphs, based on the open-source platform Apache Airflow to increase traceability and the automated execution of experiments. We demonstrate the integration and automation of experimental workflows in a laboratory environment with a heterogeneous device landscape including liquid handling stations, parallel cultivation systems, and mobile robots. The feasibility of our approach is assessed in parallel E. coli fed-batch cultivations with glucose oscillations in which different elastin-like proteins are produced. We show that the use of workflow management systems in robotic cultivation platforms increases automation, robustness and traceability of experimental data.

A workflow management system for reproducible and interoperable high-throughput self-driving experiments

To foster self-driving experimentation and address the reproducibility crisis in bioprocess development in a collaborative environment, a modular Workflow Management System (WMS) is required. In this work, a WMS based on Directed Acyclic Graphs that offers a modular and flexible design for plug-and-play integration of computational tools is presented. A case study is used to demonstrate that the implementation of a computational WMS in robotic experimental facilities promotes the application of Findable, Accessible, Interoperable and Re-usable principles, allowing researchers to readily share protocols, models, methods and data. As a proof of concept, we integrated three different computational workflows for online re-design of feeding rates in 24 parallel E. coli fed-batch cultivations producing elastin-like proteins. This approach provides a solid foundation for increasing scientific data generation in robotic experimental facilities, fostering open collaboration, and addressing the challenges of reproducibility in research.

Characterization of a Chimeric Resilin-Elastin Structural Protein Dedicated to 3D Bioprinting as a Bioink Component.

In this study we propose to use for bioprinting a bioink enriched with a recombinant RE15mR protein with a molecular weight of 26 kDa, containing functional sequences derived from resilin and elastin. The resulting protein also contains RGD sequences in its structure, as well as a metalloproteinase cleavage site, allowing positive interaction with the cells seeded on the construct and remodeling the structure of this protein in situ. The described protein is produced in a prokaryotic expression system using an E. coli bacterial strain and purified by a process using a unique combination of known methods not previously used for recombinant elastin-like proteins. The positive effect of RE15mR on the mechanical, physico-chemical, and biological properties of the print is shown in the attached results. The addition of RE15mR to the bioink resulted in improved mechanical and physicochemical properties and promoted the habitation of the prints by cells of the L-929 line.

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2–3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Engineered protein elastomeric materials.

Natural evolution endows some insects and marine organisms with a special class of protein-based elastic tissues that possess energy feedback characteristics, providing them with the foundation for jumping and flying, and protecting them from the damage caused by movements or waves. However, the design and fabrication of such protein-based elastomeric materials that can function in human society through biomimetic strategies still remains challenging. Recombinant proteins designed by synthetic biology can mimic the advantageous structures in natural proteins and can be biosynthesized without the requirements for harsh conditions such as high temperatures and cytotoxic agents, which provides a great opportunity to prepare protein-based elastomeric materials. In this review, starting from the design of protein molecules, we highlight an overview of the synthesis of elastomeric materials based on recombinant resilin, recombinant elastin-like proteins and other recombinant folded proteins, etc., and then demonstrate their application progress in the fields of biomedicine and high technology. Finally, the challenges and prospects for the future development of protein-based elastomeric materials are envisioned to provide insights into the design and synthesis of the next generation of protein-based elastomeric materials.

A Library of Elastin-like Proteins with Tunable Matrix Ligands for In Vitro 3D Neural Cell Culture.

Hydrogels with encapsulated cells have widespread biomedical applications, both as tissue-mimetic 3D cultures in vitro and as tissue-engineered therapies in vivo. Within these hydrogels, the presentation of cell-instructive extracellular matrix (ECM)-derived ligands and matrix stiffness are critical factors known to influence numerous cell behaviors. While individual ECM biopolymers can be blended together to alter the presentation of cell-instructive ligands, this typically results in hydrogels with a range of mechanical properties. Synthetic systems that allow for the facile incorporation and modulation of multiple ligands without modification of matrix mechanics are highly desirable. In the present work, we leverage protein engineering to design a family of xeno-free hydrogels (i.e., devoid of animal-derived components) consisting of recombinant hyaluronan and recombinant elastin-like proteins (ELPs), cross-linked together with dynamic covalent bonds. The ELP components incorporate cell-instructive peptide ligands derived from ECM proteins, including fibronectin (RGD), laminin (IKVAV and YIGSR), collagen (DGEA), and tenascin-C (PLAEIDGIELTY and VFDNFVL). By carefully designing the protein primary sequence, we form 3D hydrogels with defined and tunable concentrations of cell-instructive ligands that have similar matrix mechanics. Utilizing this system, we demonstrate that neurite outgrowth from encapsulated embryonic dorsal root ganglion (DRG) cultures is significantly modified by cell-instructive ligand content. Thus, this library of protein-engineered hydrogels is a cell-compatible system to systematically study cell responses to matrix-derived ligands.

Immunolocalization of Some Epidermal Proteins and Glycoproteins in the Growing Skin of the Australian Lungfish (Neoceratodus forsteri).

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as "circular bodies" in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2-3 cm in length. In the superficial dermis of juveniles (16-26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates.

Programming protein phase-separation employing a modular library of intrinsically disordered precision block copolymer-like proteins creating dynamic cytoplasmatic compartmentalization

The control of supramolecular complexes in living systems at the molecular level is an important goal in life-sciences. Spatiotemporal organization of molecular distribution & flow of such complexes are essential physicochemical processes in living cells and important for pharmaceutical processes. Membraneless organelles (MO) found in eukaryotic cells, formed by liquid-liquid phase-separation (LLPS) of intrinsically disordered proteins (IDPs) control and adjust intracellular organization. Artificially designed compartments based on LLPS open up a novel pathway to control chemical flux and partition in vitro and in vivo. We designed a library of chemically precisely defined block copolymer-like proteins based on elastin-like proteins (ELPs) with defined charge distribution and type, as well as polar and hydrophobic block domains. This enables the programmability of physicochemical properties and to control adjustable LLPS in vivo attaining control over intracellular partitioning and flux as role model for in vitro and in vivo applications. Tailor-made ELP-like block copolymer proteins exhibiting IDP-behavior enable LLPS formation in vitro and in vivo allowing the assembly of membrane-based and membraneless superstructures via protein phase-separation in E. coli. Subsequently, we demonstrate the responsiveness of protein phase-separated spaces (PPSSs) to environmental physicochemical triggers and their selective, charge-dependent and switchable interaction with DNA or extrinsic and intrinsic molecules enabling their selective shuttling across semipermeable phase boundaries including (cell)membranes. This paves the road for adjustable artificial PPSS-based storage and reaction spaces and the specific transport across phase boundaries for applications in pharmacy and synthetic biology.

Rational design of elastin-like polypeptide fusion proteins to tune self-assembly and properties of protein vesicles.

Protein vesicles made from bioactive proteins have potential value in drug delivery, biocatalysis, and as artificial cells. As the proteins are produced recombinantly, the ability to precisely tune the protein sequence provides control not possible with polymeric vesicles. The tunability and biocompatibility motivated this work to develop protein vesicles using rationally designed protein building blocks to investigate how protein sequence influences vesicle self-assembly and properties. We have reported an elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) and functional, globular proteins fused to a glutamate-rich leucine zipper (ZE) that self-assemble into protein vesicles when warmed from 4 to 25 °C due to the hydrophobic transition of ELP. Previously, we demonstrated the ability to tune vesicle properties by changing protein and salt concentration, ZE : ZR ratio, and warming rate. However, there is a limit to the properties that can be achieved via assembly conditions. In order to access a wider range of vesicle diameter and stability profiles, this work investigated how modifiying the hydrophobicity and length of the ELP sequence influenced self-assembly and the final properties of protein vesicles using mCherry as a model globular protein. The results showed that both transition temperature and diameter of protein vesicles were inversely correlated to the ELP guest residue hydrophobicity and the number of ELP pentapeptide repeats. Additionally, sequence manipulation enabled assembly of vesicles with properties not accessible by changes to assembly conditions. For example, introduction of tyrosine at 5 guest residue positions in ELP enabled formation of nanoscale vesicles stable at physiological salt concentration. This work yields design guidelines for modifying the ELP sequence to manipulate protein vesicle transition temperature, size and stability to achieve desired properties for particular biofunctional applications.

Purification of a Hydrophobic Elastin-Like Protein Toward Scale-Suitable Production of Biomaterials.

Elastin-like proteins (ELPs) are polypeptides with potential applications as renewable bio-based high-performance polymers, which undergo a stimulus-responsive reversible phase transition. The ELP investigated in this manuscript—ELP[V2Y-45]—promises fascinating mechanical properties in biomaterial applications. Purification process scalability and purification performance are important factors for the evaluation of potential industrial-scale production of ELPs. Salt-induced precipitation, inverse transition cycling (ITC), and immobilized metal ion affinity chromatography (IMAC) were assessed as purification protocols for a polyhistidine-tagged hydrophobic ELP showing low-temperature transition behavior. IMAC achieved a purity of 86% and the lowest nucleic acid contamination of all processes. Metal ion leakage did not propagate chemical modifications and could be successfully removed through size-exclusion chromatography. The simplest approach using a high-salt precipitation resulted in a 60% higher target molecule yield compared to both other approaches, with the drawback of a lower purity of 60% and higher nucleic acid contamination. An additional ITC purification led to the highest purity of 88% and high nucleic acid removal. However, expensive temperature-dependent centrifugation steps are required and aggregation effects even at low temperatures have to be considered for the investigated ELP. Therefore, ITC and IMAC are promising downstream processes for biomedical applications with scale-dependent economical costs to be considered, while salt-induced precipitation may be a fast and simple alternative for large-scale bio-based polymer production.

Dynamic Structural Changes and Thermodynamics in Phase Separation Processes of an Intrinsically Disordered-Ordered Protein Model.

Elastin‐like proteins (ELPs) are biologically important proteins and models for intrinsically disordered proteins (IDPs) and dynamic structural transitions associated with coacervates and liquid–liquid phase transitions. However, the conformational status below and above coacervation temperature and its role in the phase separation process is still elusive. Employing matrix least‐squares global Boltzmann fitting of the circular dichroism spectra of the ELPs (VPGVG)20, (VPGVG)40, and (VPGVG)60, we found that coacervation occurs sharply when a certain number of repeat units has acquired β‐turn conformation (in our sequence setting a threshold of approx. 20 repeat units). The character of the differential scattering of the coacervate suspensions indicated that this fraction of β‐turn structure is still retained after polypeptide assembly. Such conformational thresholds may also have a role in other protein assembly processes with implications for the design of protein‐based smart materials.

Elastin-like Proteins to Support Peripheral Nerve Regeneration in Guidance Conduits.

Synthetic nerve guidance conduits (NGCs) offer an alternative to harvested nerve grafts for treating peripheral nerve injury (PNI). NGCs have been made from both naturally derived and synthesized materials. While naturally derived materials typically have an increased capacity for bioactivity, synthesized materials have better material control, including tunability and reproducibility. Protein engineering is an alternative strategy that can bridge the benefits of these two classes of materials by designing cell-responsive materials that are also systematically tunable and consistent. Here, we tested a recombinantly derived elastin-like protein (ELP) hydrogel as an intraluminal filler in a rat sciatic nerve injury model. We demonstrated that ELPs enhance the probability of forming a tissue bridge between the proximal and distal nerve stumps compared to an empty silicone conduit across the length of a 10 mm nerve gap. These tissue bridges have evidence of myelinated axons, and electrophysiology demonstrated that regenerated axons innervated distal muscle groups. Animals implanted with an ELP-filled conduit had statistically higher functional control at 6 weeks than those that had received an empty silicone conduit, as evaluated by the sciatic functional index. Taken together, our data support the conclusion that ELPs support peripheral nerve regeneration in acute complete transection injuries when used as an intraluminal filler. These results support the further study of protein engineered recombinant ELP hydrogels as a reproducible, off-the-shelf alternative for regeneration of peripheral nerves.

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging. Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Bioengineered elastin- and silk-biomaterials for drug and gene delivery.

Advances in medical science have led to diverse new therapeutic modalities, as well as enhanced understanding of the progression of various disease states. These findings facilitate the design and development of more customized and exquisite drug delivery systems that aim to improve therapeutic indices of drugs to treat a variety of conditions. Synthetic polymer-based drug carriers have often been the focus of such research. However, these structures suffer from challenges with heterogeneity of the starting material, limited chemical features, complex functionalization methods, and in some cases a lack of biocompatibility. Consequently, protein-based polymers have garnered much attention in recent years due to their monodisperse features, ease of production and functionalization, and biocompatibility. Genetic engineering techniques enable the advancement of protein-based drug delivery systems with finely tuned physicochemical properties, and thus an expanded level of customization unavailable with synthetic polymers. Of these genetically engineered proteins, elastin-like proteins (ELP), silk-like proteins (SLP), and silk-elastin-like proteins (SELP) provide a unique set of alternatives for designing drug delivery systems due to their inherent chemical and physical properties and ease of engineering afforded by recombinant DNA technologies. In this review we examine the advantages of genetically engineered drug delivery systems with emphasis on ELP and SLP constructions. Methods for fabrication and relevant biomedical applications will also be discussed.

Targeted Elastin-like Fusion Proteins for Enhanced Imaging and Treatment of Non-Muscle Invasive Bladder Cancer

Background and Hypothesis: A recent study estimates 17,670 individuals will die from bladder cancer in the United States in 2019. With current imaging and treatment technology, bladder cancer is associated with high recurrence rates making it the highest lifetime treatment cost per patient. High expense is primarily due to the profound heterogeneity associated with bladder cancer necessitating repeated intervention. Traditional treatment of non-muscle invasive bladder (NMIBC) cancer following intravesical tumor resection often includes chemotherapy or immunostimulatory therapy via Bacillus Calmette-Guerin (BCG) infusions. Both lack specificity for cancerous tissue, leading to sub-optimal prognosis and unnecessary discomfort for patients. Furthermore, bladder cancer detection is limited in its precision. Methods such as CT/MRI urography and cystoscopies are unable to accurately detect tumors under 2 centimeters in diameter. Blue light cystoscopy, a gold standard detection method, is unable to accurately differentiate tumors from inflamed tissue. This leads to issues detecting aggressive carcinoma in situ. The goal of this project is to enhance tumor detection by fusing various targeting ligands to elastin-like peptides (ELP).
 Experimental Design or Project Methods: Using synthetic biology approaches, a library of ELP fusions were made and purified using a simplified organic extraction method. (Biomaterials Science 2018, 6 (4), 863-876) This resulted in exceptionally pure protein within a matter of hours. Contrast agents were covalently attached, and performance testing was done in murine bladder cancer cells.
 Results: Novel ELP fusions were successfully produced and sequences verified. Protein expression and purification yielded active targeted constructs which showed enhanced binding efficiency over non-targeted constructs. These ELP fusion proteins are effective transporters for rapid and preferential cargo entry into bladder cancer tissue.
 Conclusion and Potential Impact: Chemotherapeutic, immunostimulatory, and tumor imaging cargo are all bright candidates for targeted ELP fusion technology. Targeted ELP delivery may be a superior tool key to improving tumor visualization and treatment, leading to improved comfort and prognosis in patients with NMIBC.

Minimalist Protocell Design: A Molecular System Based Solely on Proteins that Form Dynamic Vesicular Membranes Embedding Enzymatic Functions.

Life in its molecular context is characterized by the challenge of orchestrating structure, energy and information processes through compartmentalization and chemical transformations amenable to mimicry of protocell models. Here we present an alternative protocell model incorporating dynamic membranes based on amphiphilic elastin-like proteins (ELPs) rather than phospholipids. For the first time we demonstrate the feasibility of combining vesicular membrane formation and biocatalytic activity with molecular entities of a single class: proteins. The presented self-assembled protein-membrane-based compartments (PMBCs) accommodate either an anabolic reaction, based on free DNA ligase as an example of information transformation processes, or a catabolic process. We present a catabolic process based on a single molecular entity combining an amphiphilic protein with tobacco etch virus (TEV) protease as part of the enclosure of a reaction space and facilitating selective catalytic transformations. Combining compartmentalization and biocatalytic activity by utilizing an amphiphilic molecular building block with and without enzyme functionalization enables new strategies in bottom-up synthetic biology, regenerative medicine, pharmaceutical science and biotechnology.

Self-Assembly Toolbox of Tailored Supramolecular Architectures Based on an Amphiphilic Protein Library.

The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self-assembly of supramolecular structures with desired shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self-assembly of tailored amphiphilic elastin-like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced. The described assembly protocols efficiently yield protein membrane-based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.

Impact of Elastin-like Protein Temperature Transition on PEG-ELP Hybrid Hydrogel Properties.

Biomaterial hydrogels are made by cross-linking either natural materials, which exhibit inherent bioactivity but suffer from batch-to-batch variations, or synthetic materials, which have a well-defined chemical structure but usually require chemical modification to exhibit bioactivity. Recombinant engineered proteins bridge the divide between natural and synthetic materials because proteins incorporate bioactive domains within the biopolymer backbone and have a well-defined amino acid structure and sequence. Recombinant engineered elastin-like proteins (ELPs) are modeled from the native tropoelastin sequence. ELPs are composed of repeating VPGxG penta-peptide sequences, where x is any guest residue except proline. ELPs undergo a lower critical solution temperature (LCST) transition above which they aggregate into a coacervate phase in aqueous solution. Here we show that the LCST transition impacts hydrogel microarchitecture which may serve as a useful design feature in engineering ELP-based hydrogels. We investigate how the ELP LCST transition contributes to the properties of hybrid poly(ethylene glycol) (PEG) and ELP (PEG-ELP) hydrogels. PEG-ELP hydrogels gelled below the LCST have a homogeneous distribution of ELP, while gelling above the LCST results in the formation of spherical ELP-rich regions within the bulk hydrogel. The ELP-rich microarchitecture is maintained when an amine-reactive cross-linker is incorporated during the gelation process. The formation of ELP-rich regions reduces PEG-ELP hydrogel bulk stiffness and increases optical density. Our characterizations of hydrogels created by using the LCST transition provide design criteria for incorporating microscale features. This may be a useful technique in understanding the role of localized bioactivity at the microscale level within hydrogel systems.