Elastin-like Protein Research Articles

A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification

Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein-ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.

Tetrakis(hydroxymethyl) Phosphonium Chloride as a Covalent Cross-Linking Agent for Cell Encapsulation within Protein-Based Hydrogels

Native tissues provide cells with complex, three-dimensional (3D) environments comprised of hydrated networks of extracellular matrix proteins and sugars. By mimicking the dimensionality of native tissue while deconstructing the effects of environmental parameters, protein-based hydrogels serve as attractive, in vitro platforms to investigate cell-matrix interactions. For cell encapsulation, the process of hydrogel formation through physical or covalent cross-linking must be mild and cell compatible. While many chemical cross-linkers are commercially available for hydrogel formation, only a subset are cytocompatible; therefore, the identification of new and reliable cytocompatible cross-linkers allows for greater flexibility of hydrogel design for cell encapsulation applications. Here, we introduce tetrakis(hydroxymethyl) phosphonium chloride (THPC) as an inexpensive, amine-reactive, aqueous cross-linker for 3D cell encapsulation in protein-based hydrogels. We characterize the THPC-amine reaction by demonstrating THPC's ability to react with primary and secondary amines of various amino acids. In addition, we demonstrate the utility of THPC to tune hydrogel gelation time (6.7±0.2 to 27±1.2 min) and mechanical properties (storage moduli ∼250 Pa to ∼2200 Pa) with a recombinant elastin-like protein. Lastly, we show cytocompatibility of THPC for cell encapsulation with two cell types, embryonic stem cells and neuronal cells, where cells exhibited the ability to differentiate and grow in elastin-like protein hydrogels. The primary goal of this communication is to report the identification and utility of tetrakis(hydroxymethyl) phosphonium chloride (THPC) as an inexpensive but widely applicable cross-linker for protein-based materials.

ELP-z and ELP-zz capturing scaffolds for the purification of immunoglobulins by affinity precipitation

The increasing demand of monoclonal antibodies for therapeutic applications along with the high manufacturing cost have made it necessary to evaluate better process options and technologies for their purification. Affinity precipitation is an attractive alternative to traditional chromatographic methods by affording effective purification using a simple environmental trigger. The feature of elastin-like-protein (ELP) fused with antibody binding domains has already been explored for the purification of antibodies. However, ELP when fused with the bulkier domains such as Protein A, resulted in lower protein production. In this study, ELP was fused to smaller synthetic IgG binding domains such as the z or zz domain, resulting in up to 10-fold higher level of production. Both ELP-z and ELP-zz bind tightly to human immunoglobulin (HIgG) with a dissociation constant of 768±142nM and 68±23nM, respectively. Owing to the higher binding affinity, the use of ELP-zz resulted in more than 99% recovery of HIgG in four repeated binding and elution cycles with no observable decrease in the purification performance. The same binding and elution cycle was successfully implemented for the purification of monoclonal antibodies from hybridoma culture supernatant with close to 100% recovery.

Human recombinant elastin-like protein coatings for muscle cell proliferation and differentiation

Recombinant proteins represent a new and promising class of polymeric materials in the field of biomaterials research. An important model for biomaterial design is elastin, the protein accounting for the elasticity of several tissues. Human elastin-like polypeptides (HELPs) have been developed as recombinant versions of elastin with the purpose of enhancing some peculiar characteristics of the native protein, like self-assembling. In this paper, we report on a comparative study of rat myoblasts response to coatings based on two different HELP macromolecules, with respect to control cultures on bare cell culture polystyrene and on a standard collagen coating. Cell behavior was analyzed in terms of adhesion, proliferation and differentiation. The collected data strongly suggest the use of HELPs as excellent biomaterials for tissue engineering and regenerative medicine applications.

Sequence-specific crosslinking of electrospun, elastin-like protein preserves bioactivity and native-like mechanics.

A nanoscale mimic of the extracellular matrix is electrospun from a highly tunable family of elastin-like proteins. A sequence-specific, two-step crosslinking procedure is developed to preserve the nanofiber morphology, elastin-like mechanics, and specific bioactivity. Rodent marrow stromal cells show sequence-specific adhesion on the matrices, which are imaged using label-free coherent anti-Stokes Raman scattering (CARS) microscopy.

ChemInform Abstract: Recombinant Protein‐Based Polymers for Advanced Drug Delivery

AbstractReview: 95 refs.

Engineering a high‐affinity scaffold for non‐chromatographic protein purification via intein‐mediated cleavage

While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale-up have promoted the development of alternative non-chromatographic separation methods. Here we developed a new non-chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo-responsive elastin-like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin-dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used.

Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings.

Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.

Recombinant protein-based polymers for advanced drug delivery

Advances in recombinant techniques have led to the development of genetically engineered polymers with exquisite control over monomer sequence and polymer length. The ability to study how precise structures correlate with function has provided opportunities for the utility of these polymers in drug delivery. Chemically derived and developed methods of synthesis have yielded many useful polymers for drug delivery to-date, including those currently used in patients. However they have drawbacks, including limitations involved in statistical characterization of conventional polymer synthetic techniques. Encoding at the genetic level and production of such recombinant polymers in organisms allow for precise order and accuracy of amino acid residues and production of monodisperse polymers with specific function and physicochemical properties. Research into elastin-like, silk-like, and silk-elastinlike protein polymers for example has led to the development of delivery systems based on natural motifs of structural proteins to take advantage of their physicochemical properties. Additionally, protein based polymers on other natural motifs and de novo designs are starting to produce promising constructs for drug and gene delivery applications where precise control over structure promises correlation with function and guides the development of new and improved constructs. Clinical applications based on recombinant polymers for delivery of bioactive agents have not been realized at this point. However lessons learned from fundamental research with these polymers can be used to guide design of safe and effective systems for use in the clinic. This tutorial review summarizes progress made in the design and utility of recombinant polymers in drug and gene delivery and discusses challenges and future directions of such polymers for this purpose.

Hydrazone self-crosslinking of multiphase elastin-like block copolymer networks

Biosynthetic strategies for the production of recombinant elastin-like protein (ELP) triblock copolymers have resulted in elastomeric protein hydrogels, formed through rapid physical crosslinking upon warming of concentrated solutions. However, the strength of physically crosslinked networks can be limited, and options for non-toxic chemical crosslinking of these networks are not optimal. In this report, we modify two recombinant elastin-like proteins with aldehyde and hydrazide functionalities. When combined, these modified recombinant proteins self-crosslink through hydrazone bonding without requiring initiators or producing by-products. Crosslinked materials are evaluated for water content and swelling upon hydration, and subject to tensile and compressive mechanical tests. Hydrazone crosslinking is a viable method for increasing the mechanical strength of elastin-like protein polymers, in a manner that is likely to lend itself to the biocompatible in situ formation of chemically and physically crosslinked ELP hydrogels.

Maleimide–thiol coupling of a bioactive peptide to an elastin-like protein polymer

Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.

Tunable Self-Assembly of Genetically Engineered Silk–Elastin-like Protein Polymers

Silk--elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Because of the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk-to-elastin blocks in a SELP's repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels, and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering, and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.

SoxE gene duplication and development of the lamprey branchial skeleton: Insights into development and evolution of the neural crest

SoxE genes are multifunctional transcriptional regulators that play key roles in specification and differentiation of neural crest. Three members (Sox8, Sox9, Sox10) are expressed in the neural crest and are thought to modulate the expression and activity of each other. In addition to regulating the expression of other early neural crest marker genes, SoxE genes are required for development of cartilage. Here we investigated the role of SoxE genes in development of the neural crest-derived branchial skeleton in the sea lamprey. Using a morpholino knockdown approach, we show that all three SoxE genes described in lamprey are required for branchial basket development. Our results suggest that SoxE1 and SoxE2 are required for specification of the chondrogenic neural crest. SoxE3 plays a morphogenetic role in patterning of the branchial basket and may be required for the development of mucocartilage, a tissue unique to larval lampreys. While the lamprey branchial basket develops primarily from an elastin-like major extracellular matrix protein that is specific to lampreys, fibrillar collagen is also expressed in developing branchial cartilage and may be regulated by the lamprey SoxE genes. Our data suggest that the regulation of Type II collagen by Sox9 might have been co-opted by the neural crest in development of the branchial skeleton following the divergence of agnathan and gnathostome vertebrates. Finally, our results also have implications for understanding the independent evolution of duplicated SoxE genes among agnathan and gnathostome vertebrates.

Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair

Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23–314%), six-fold in Young’s modulus (5.3–33.1 MPa), and more than two-fold in tensile strength (1.85–4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity.

A smart bilayer scaffold of elastin-like recombinamer and collagen for soft tissue engineering

Elastin-like recombinamers (ELRs) are smart, protein-based polymers designed with desired peptide sequences using recombinant DNA technology. The aim of the present study was to produce improved tissue engineering scaffolds from collagen and an elastin-like protein tailored to contain the cell adhesion peptide RGD, and to investigate the structural and mechanical capacities of the resulting scaffolds (foams, fibers and foam-fiber bilayer scaffolds). The results of the scanning electron microscopy, mercury porosimetry and mechanical testing indicated that incorporation of ELR into the scaffolds improved the uniformity and continuity of the pore network, decreased the pore size (from 200 to 20 μm) and the fiber diameter (from 1.179 μm to 306 nm), broadened the pore size distribution (from 70-200 to 4-200 μm) and increased their flexibility (from 0.007 to 0.011 kPa⁻¹). Culture of human fibroblasts and epithelial cells in ELR-collagen scaffolds showed the positive contribution of ELR on proliferation of both types of cells.

The use of microfiber composites of elastin-like protein matrix reinforced with synthetic collagen in the design of vascular grafts

Collagen and elastin networks contribute to highly specialized biomechanical responses in numerous tissues and species. Biomechanical properties such as modulus, elasticity, and strength ultimately affect tissue function and durability, as well as local cellular behavior. In the case of vascular bypass grafts, compliance at physiologic pressures is correlated with increased patency due to a reduction in anastomotic intimal hyerplasia. In this report, we combine extracellular matrix (ECM) protein analogues to yield multilamellar vascular grafts comprised of a recombinant elastin-like protein matrix reinforced with synthetic collagen microfibers. Structural analysis revealed that the fabrication scheme permits a range of fiber orientations and volume fractions, leading to tunable mechanical properties. Burst strengths of 239–2760 mm Hg, compliances of 2.8–8.4%/100 mm Hg, and suture retention strengths of 35–192 gf were observed. The design most closely approximating all target criteria displayed a burst strength of 1483 ± 143 mm Hg, a compliance of 5.1 ± 0.8%/100 mm Hg, and a suture retention strength of 173 ± 4 gf. These results indicate that through incorporation of reinforcing collagen microfibers, recombinant elastomeric protein-based biomaterials can play a significant role in load bearing tissue substitutes. We believe that similar composites can be incorporated into tissue engineering schemes that seek to integrate cells within the structure, prior to or after implantation in vivo.

Microcrimped Collagen Fiber‐Elastin Composites

A flexible membrane templating technique (a) is developed for the fabrication of microcrimped collagen microfibers (b) that are embedded in an elastin-like protein matrix to generate hierarchical, biologically inspired fiber-reinforced composites (c). The microcrimped structure is stable under cyclic loading and the mechanical response of the engineered collagen-elastin composite mimics that of native tissue.

Expression of a recombinant elastin‐like protein in pichia pastoris

The translation of highly repetitive gene sequences is often associated with reduced levels of protein expression and may be prone to mutational events. In this report, we describe a modified concatemerization strategy to construct a gene with enhanced sequence diversity that encodes a highly repetitive elastin-like protein polymer for expression in Pichia pastoris. Specifically, degenerate oligonucleotides were used to create a monomer library, which after concatemerization yielded a genetically nonrepetitive DNA sequence that encoded identical pentapeptide repeat sequences. By limiting genetic repetition, the risk of genetic deletions, rearrangements, or premature termination errors during protein synthesis is minimized.

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions.

BackgroundElastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants.ResultsThe effect of elastin-like polypeptide fusions on the accumulation of green fluorescent protein targeted to the cytoplasm, chloroplasts, apoplast, and endoplasmic reticulum was evaluated. The endoplasmic reticulum was the only intracellular compartment in which an elastin-like polypeptide tag was shown to significantly enhance recombinant protein accumulation. Interestingly, endoplasmic reticulum-targeted elastin-like polypeptide fusions induced the formation of a novel type of protein body, which may be responsible for elastin-like polypeptide's positive effect on recombinant protein accumulation by excluding the heterologous protein from normal physiological turnover. Although expressed in the leaves of plants, these novel protein bodies appeared similar in size and morphology to the prolamin-based protein bodies naturally found in plant seeds. The elastin-like polypeptide-induced protein bodies were highly mobile organelles, exhibiting various dynamic patterns of movement throughout the cells, which were dependent on intact actin microfilaments and a functional actomyosin motility system.ConclusionAn endoplasmic reticulum-targeted elastin-like polypeptide fusion approach provides an effective strategy for depositing large amounts of concentrated heterologous protein within the limited space of the cell via storage in stable protein bodies. Furthermore, encapsulation of recombinant proteins into physiologically inert organelles can function to insulate the protein from normal cellular mechanisms, thus limiting unnecessary stress to the host cell. Since elastin-like polypeptide is a mammalian-derived protein, this study demonstrates that plant seed-specific factors are not required for the formation of protein bodies in vegetative plant tissues, suggesting that the endoplasmic reticulum possesses an intrinsic ability to form protein body-like accretions in eukaryotic cells when overexpressing particular proteins.

Dynamic cell culturing and its application to micropatterned, elastin-like protein-modified poly( N-isopropylacrylamide) scaffolds

In this study a tissue engineering scaffold was constructed from poly( N-isopropylacrylamide) (pNIPAM) to study the influence of strain on cell proliferation and differentiation. The effect of surface chemistry and topography on bone marrow mesenchymal stem cells was also investigated. Micropatterned pNIPAM films (channels with 10 μm groove width, 2 μm ridge width, 20 μm depth) were prepared by photopolymerization. The films were chemically modified by adsorption of a genetically engineered and temperature sensitive elastin-like protein (ELP). Dynamic conditions were generated by repeated temperature changes between 29 °C and 37 °C. ELP presence on the films enhanced initial cell attachment two fold (Day 1 cell number on films with ELP and without ELP were 27.6 × 10 4 and 13.2 × 10 4, respectively) but had no effect on proliferation in the long run. ELP was crucial for maintaining the cells attached on the surface in dynamic culturing (Day 7 cell numbers on the films with and without ELP were 81.4 × 10 4 and 12.1 × 10 4, respectively) and this enhanced the ability of pNIPAM films to transfer mechanical stress on the cells. Dynamic conditions improved cell proliferation (Day 21 cell numbers with dynamic and with static groups were 180.4 × 10 4 and 157.7 × 10 4, respectively) but decreased differentiation (Day 14 specific ALP values on the films of static and dynamic groups were 6.6 and 3.5 nmol/min/cell, respectively). Thus, a physically and chemically modified pNIPAM scaffold had a positive influence on the population of the scaffolds under dynamic culture conditions.