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Protein purification is a crucial step for various downstream applications like drug development, antibody preparation, and structure determination. The constant pursuit is for methods that are more efficient and cost-effective. We propose a novel approach using an elastin-like polypeptide (ELP) as an aggregation core that serves as an anchor between the beads in a chromatography column. In this method, a chilled sample containing a [target protein type] fusion protein is loaded onto a pre-equilibrated IMAC (immobilized metal affinity chromatography) column with a low-salt buffer. The column is then washed with a warm buffer containing high salt to remove impurities. Here, the key step involves warming the column above the ELP's transition temperature (Tt), which triggers its aggregation. This aggregation is expected to trap the target protein tightly between the beads. Subsequently, a harsh wash with high salt and high imidazole can be applied to remove even persistent contaminants, achieving high protein purity. Finally, the temperature is lowered, and a cold, low-salt buffer is introduced to reverse the aggregation and elute the purified target protein. This method has the potential to eliminate the need for sophisticated chromatography systems while still achieving high protein purity. [ABSTRACT FROM AUTHOR]

(1) Background: This study aimed to enhance the biological properties of hydraulic calcium silicate cements (HCSCs) by incorporating organic and inorganic components, specifically elastin-like polypeptides (ELPs) and bioactive glass (BAG). We focused on the effects of these composites on the viability, migration, and osteogenic differentiation of human periodontal ligament fibroblasts (hPDLFs). (2) Methods: Proroot MTA was supplemented with 1–5 wt% 63S BAG and 10 wt% ELP. The experimental groups contained various combinations of HSCS with ELP and BAG. Cell viability was assessed using an MTT assay, cell migration was evaluated using wound healing and transwell assays, and osteogenic activity was determined through Alizarin Red S staining and a gene expression analysis of osteogenic markers (ALP, RUNX-2, OCN, and Col1A2). (3) Results: The combination of ELP and BAG significantly enhanced the viability of hPDLFs with an optimal BAG concentration of 1–4%. Cell migration assays demonstrated faster migration rates in groups with 2–4% BAG and ELP incorporation. Osteogenic activity was the highest with 2–3% BAG incorporation with ELP, as evidenced by intense Alizarin Red S staining and the upregulation of osteogenic differentiation markers. (4) Conclusions: The incorporation of ELP (organic) and BAG (inorganic) into HCSC significantly enhances the viability, migration, and osteogenic differentiation of hPDLFs. These findings suggest that composite HCSC might support healing in destructed bone lesions in endodontics. [ABSTRACT FROM AUTHOR]

Temperature-responsive elastin-like polypeptides (ELPs) exhibit a low critical solution temperature-type phase transition and offer potential as useful materials for the construction of nanoparticles. Herein, we developed a novel decoration method for ELP-based nanoparticles via isopeptide bond formation with the SnoopTag/SnoopCatcher system that is not affected by the heating process required for particle formation. A mixture of a fusion protein of ELP and poly(aspartic acid) (poly(D)), known as ELP-poly(D), and ELP-poly(D) fused with SnoopCatcher (ELP-poly(D)-SnC) formed protein nanoparticles as a result of the temperature responsiveness of ELP, with the resultant nanoparticles displaying the SnoopCatcher binding domain on their surfaces. In the present study, two model proteins fused to SnoopTag were displayed on the surfaces of protein nanoparticles constructed from ELP-poly(D)-SnC and ELP-poly(D). The model proteins are enhanced green fluorescent protein (EGFP) and Renilla luciferace (Rluc), which exhibits luminescent capability and weak thermostability, respectively. EGFP on the particle surface was found to retain 48.7% activity, while Rluc exhibited almost full activity, as calculated from the binding efficiency and nanoparticle activities recovered after purification. ELP-based nanoparticles containing the SnoopTag/SnoopCatcher system offer the opportunity for particle decoration with a wide range of functional proteins via isopeptide bond formation. [ABSTRACT FROM AUTHOR]

Elastin‐like polypeptide (ELP) is a thermo‐sensitive biosynthetic polymer composed of a Val‐Pro‐Gly‐Xaa‐Gly repeating unit possessing a sharp reversible phase transition property at specific temperatures. Here, ELP was fused to human superoxide dismutase 1 (hSOD1) modified with His tag to produce recombinant hSOD1‐Linker‐ELP‐His (hSODLEH) which was expressed in E. coli and purified via inverse thermal cycling (ITC) and Ni‐NTA resin. The results showed that ELP tag did not affect the soluble expression of SOD1. The purification by ITC was superior to Ni‐NTA resin due to its convenient purification process, improved recovery rate and purification fold, thus indicating industrial suitability for large scale protein purification in a cost‐ and time‐efficient manner. Moreover, one round of ITC was sufficient for the recovery of highly purified recombinant SOD. The results further showed that ELP improved the stability of the SOD, and did not affect its secondary structure nor its ability to bind divalent metal ions. Overall, ELP‐protein fusion system could be a promising tool for large scale enzyme purification. [ABSTRACT FROM AUTHOR]

A generic model of elastin–like polypeptides (ELP) is derived that includes proline isomerization (ProI). As a case study, conformational transition of a –[valine–proline–glycine–valine–glycine]– sequence is investigated in aqueous ethanol mixtures. While the non–bonded interactions are based on the Lennard–Jones (LJ) parameters, the effect of ProI is incorporated by tuning the intramolecular 3– and 4–body interactions known from the underlying all–atom simulations into the generic model. One of the key advantages of such a minimalistic model is that it readily decouples the effects of geometry and the monomer–solvent interactions due to the presence of ProI, thus gives a clearer microscopic picture that is otherwise rather nontrivial within the all–atom setups. These results are consistent with the available all–atom and experimental data. The model derived here may pave the way to investigate large scale self–assembly of ELPs or biomimetic polymers in general. [ABSTRACT FROM AUTHOR]

Successful nerve repair using bioadhesive hydrogels demands minimizing tissue–material interfacial mechanical mismatch to reduce immune responses and scar tissue formation. Furthermore, it is crucial to maintain the bioelectrical stimulation‐mediated cell‐signaling mechanism to overcome communication barriers within injured nerve tissues. Therefore, engineering bioadhesives for neural tissue regeneration necessitates the integration of electroconductive properties with tissue‐like biomechanics. In this study, we propose a stretchable bioadhesive based on a custom‐designed chemically modified elastin‐like polypeptides (ELPs) and a choline‐based bioionic liquid (Bio‐IL), providing an electroconductive microenvironment to reconnect damaged nerve tissue. The stretchability akin to native neural tissue was achieved by incorporating hydrophobic ELP pockets, and a robust tissue adhesion was obtained due to multi‐mode tissue–material interactions through covalent and noncovalent bonding at the tissue interface. Adhesion tests revealed adhesive strength ~10 times higher than commercially available tissue adhesive, Evicel®. Furthermore, the engineered hydrogel supported in vitro viability and proliferation of human glial cells. We also evaluated the biodegradability and biocompatibility of the engineered bioadhesive in vivo using a rat subcutaneous implantation model, which demonstrated facile tissue infiltration and minimal immune response. The outlined functionalities empower the engineered elastic and electroconductive adhesive hydrogel to effectively enable sutureless surgical sealing of neural injuries and promote tissue regeneration. [ABSTRACT FROM AUTHOR]

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models. [ABSTRACT FROM AUTHOR]

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Elastin-like polypeptides (ELPs) are thermally responsive biopolymers derived from natural elastin. These peptides have a low critical solution temperature phase behavior and can be used to prepare stimuli-responsive biomaterials. Through genetic engineering, biomaterials prepared from ELPs can have unique and customizable properties. By adjusting the amino acid sequence and length of ELPs, nanostructures, such as micelles and nanofibers, can be formed. Correspondingly, ELPs have been used for improving the stability and prolonging drug-release time. Furthermore, ELPs have widespread use in tissue repair due to their biocompatibility and biodegradability. Here, this review summarizes the basic property composition of ELPs and the methods for modulating their phase transition properties, discusses the application of drug delivery system and tissue repair and clarifies the current challenges and future directions of ELPs in applications. [ABSTRACT FROM AUTHOR]

L‐Asparaginase (ASP) is well‐known for its excellent efficacy in treating hematological malignancies. Unfortunately, the intrinsic shortcomings of ASP, namely high immunogenicity, severe toxicity, short half‐life, and poor stability, restrict its clinical usage. Poly(ethylene glycol) conjugation (PEGylation) of ASP is an effective strategy to address these issues, but it is not ideal in clinical applications due to complex chemical synthesis procedures, reduced ASP activity after conjugation, and pre‐existing anti‐PEG antibodies in humans. Herein, the authors genetically engineered an elastin‐like polypeptide (ELP)‐fused ASP (ASP‐ELP), a core‐shell structured tetramer predicted by AlphaFold2, to overcome the limitations of ASP and PEG‐ASP. Notably, the unique thermosensitivity of ASP‐ELP enables the in situ formation of a sustained‐release depot post‐injection with zero‐order release kinetics over a long time. The in vitro and in vivo studies reveal that ASP‐ELP possesses increased activity retention, improved stability, extended half‐life, mitigated immunogenicity, reduced toxicity, and enhanced efficacy compared to ASP and PEG‐ASP. Indeed, ASP‐ELP treatment in leukemia or lymphoma mouse models of cell line‐derived xenograft (CDX) shows potent anti‐cancer effects with significantly prolonged survival. The findings also indicate that artificial intelligence (AI)‐assisted genetic engineering is instructive in designing protein‐polypeptide conjugates and may pave the way to develop next‐generation biologics to enhance cancer treatment. [ABSTRACT FROM AUTHOR]

Strategic materials design is essential for the development of small-diameter, tissue-engineered vascular grafts. Self-assembled nanofibers of elastin-like polypeptides represent promising vascular graft components as they replicate the organized elastin structure of native blood vessels. Further, the bioactivity of nanofibers can be modified by the addition of functional peptide motifs. In the present study, we describe the development of a novel nanofiber-forming elastin-like polypeptide (ELP) with an arginine–glutamic acid–aspartic acid–valine (REDV) sequence. The biological characteristics of the REDV-modified ELP nanofibers relevant to applications in vascular grafting were compared to ELP without ligands for integrin, ELP with arginine–glycine–aspartic acid (RGD) sequence, collagen and cell culture glass. Among them, REDV-modified ELP nanofibers met the preferred biological properties for vascular graft materials, i.e. (i) inhibition of platelet adhesion and activation, (ii) endothelial cell adhesion and proliferation and (iii) maintenance of smooth muscle cells in a contractile phenotype to prevent cell overgrowth. The results indicate that REDV-modified ELP nanofibers represent promising candidates for the further development of small-diameter vascular grafts. [ABSTRACT FROM AUTHOR]

There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of selfassembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 μg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT. [ABSTRACT FROM AUTHOR]

Proliferative Vitreoretinopathy (PVR) is a refractory retinal disease whose primary pathogenesis involves the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. At present, there is no effective treatment other than surgery for PVR. The purpose of this study was to investigate the effect of αB crystallin peptide (αBC-P) on EMT in PVR. We have previously shown that this peptide is antiapoptotic and regulates RPE redox status. Subconfluent primary human RPE (hRPE) cells were stimulated by TGFβ2 (10 ng/mL) with or without αBC-P (50 or 75 μg/mL) for 48 h and expression of EMT/mesenchymal to epithelial transition (MET) markers was determined. Mitochondrial ROS (mtROS) generation in hRPE cells treated with TGFβ2 was analyzed. The effect of TGFβ2 and αBC-P on oxidative phosphorylation (OXPHOS) and glycolysis in hRPE was studied. RPE cell migration was also assessed. A PVR-like phenotype was induced by intravitreal dispase injection in C57BL/6J mice. PVR progression and potential therapeutic efficiency of αBC-Elastin-like polypeptides (ELP) was studied using fundus photography, OCT imaging, ERG, and histologic analysis of the retina. αSMA, E-cadherin, Vimentin, Fibronectin and, RPE65, and CTGF were analyzed on Day 28. Additionally, the amount of VEGF-A in retinal cell lysates was measured. The EMT-associated αSMA, Vimentin, SNAIL and SLUG showed a significant upregulation with TGFβ2, and their expression was significantly suppressed by cotreatment with αBC-P. The MET-associated markers, E-cadherin and Sirt1, were significantly downregulated by TGFβ2 and were restored by αBC-P. Incubation of hRPE with TGFβ2 for 24 h showed a marked increase in mitochondrial ROS which was noticeably inhibited by αBC-ELP. We also showed that after TGFβ2 treatment, SMAD4 translocated to mitochondria which was blocked by αBC-ELP. Mitochondrial oxygen consumption rate increased with TGFβ2 treatment for 48 h, and αBC-P co-treatment caused a further increase in OCR. Glycolytic functions of RPE were significantly suppressed with αBC-P (75 μg/mL). In addition, αBC-P significantly inhibited the migration from TGFβ2 treatment in hRPE cells. The formation of proliferative membranes was suppressed in the αBC-ELP-treated group, as evidenced by fundus, OCT, and H&E staining in dispase-induced PVR in mice. Furthermore, ERG showed an improvement in c-wave amplitude. In addition, immunostaining showed significant suppression of αSMA and RPE65 expression. It was also observed that αBC-ELP significantly reduced the expression level of vimentin, fibronectin, and CTGF. Our findings suggest that the antioxidant αBC-P may have therapeutic potential in preventing PVR by reversing the phenotype of EMT/MET and improving the mitochondrial function in RPE cells. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is a primary brain tumor that carries a dismal prognosis, which is primarily attributed to tumor recurrence after surgery and resistance to chemotherapy. Since the tumor recurrence appears near the site of surgical resection, a concept of immediate and local application of chemotherapeutic after initial tumor removal could lead to improved treatment outcome. With the ultimate goal of developing a locally-applied, injectable drug delivery vehicle for GBM treatment, we created elastin-like polypeptide (ELP) hydrogels. The ELP hydrogels can be engineered to release anti-cancer drugs over an extended period. The purpose of this study was to evaluate the biomechanical properties of ELP hydrogels, to characterize their ability to release doxorubicin over time, and to investigate, in vitro, the anti-proliferative effect of Dox-laden ELP hydrogels on GBM. Here, we present microstructural differences, swelling ratio measurements, drug release characteristics, and in vitro effects of different ELP hydrogel compositions. We found that manipulation of the ELP–collagen ratio allows for tunable drug release, that the released drug is taken up by cells, and that incubation with a small volume of ELP-Dox hydrogel drastically reduced survival and proliferation of GBM cells in vitro. These results underscore the potential of ELP hydrogels as a local delivery strategy to improve prognosis for GBM patients after tumor resection. [ABSTRACT FROM AUTHOR]

The anticancer agent doxorubicin(dox) has been widely used in the treatment of a variety of hematological malignancies and solid tumors. Despite doxorubicin's efficiency in killing tumor cells, severe damage to healthy tissues, along with cardiotoxicity, limits its clinical use. To overcome these adverse side effects, improve patient safety, and enhance therapeutic efficacy, we have designed a thermally responsive biopolymer doxorubicin carrier that can be specifically targeted to tumor tissue by locally applying mild hyperthermia (41 °C). The developed drug vehicle is composed of the following: a cell penetrating peptide (SynB1) to promote tumor and cellular uptake; thermally responsive Elastin-like polypeptide (ELP); and the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) containing a pH-sensitive hydrazone linker that releases doxorubicin in the acidic tumor environment. We used the in vivo imaging system, IVIS, to determine biodistribution of doxorubicin-delivered ELP in MDA-MB-231 xenografts in nude mice. Tumor bearing mice were treated with a single IV injection of 10 mg/kg doxorubicin equivalent dose with free doxorubicin, thermally responsive SynB1 ELP 1-DOXO, and a thermally nonresponsive control biopolymer, SynB1 ELP 2-DOXO. Following a 2 h treatment with hyperthermia, tumors showed a 2-fold higher uptake when treated with SynB1 ELP 1-DOXO compared to free doxorubicin. Accumulation of the thermally non-responsive control SynB1 ELP2 –DOXO was comparable to free doxorubicin, indicating that an increase in dox accumulation with ELP is due to aggregation in response to thermal targeting. Higher levels of SynB1 ELP1–DOXO and SynB1 ELP2 –DOXO with respect to free doxorubicin were observed in kidneys. Fluorescence intensity from hearts of animals treated with SynB1 ELP1–DOXO show a 5-fold decrease in accumulation of doxorubicin than the same dose of free doxorubicin. SynB1-ELP1-DOXO biopolymers demonstrated a 6-fold increase in tumor/heart ratio in comparison to free doxorubicin, indicating preferential accumulation of the drug in tumors. These results demonstrate that thermally targeted polymers are a promising therapy to enhance tumor targeting and uptake of anticancer drugs and to minimize free drug toxicity in healthy tissues, representing a great potential for clinical application. [ABSTRACT FROM AUTHOR]

Regenerative medicine using stem cells offers promising strategies for treating a variety of degenerative diseases. Regulation of stem cell behavior and rejuvenate senescence are required for stem cells to be clinically effective. The extracellular matrix (ECM) components have a significant impact on the stem cell's function and fate mimicking the local environment to maintain cells or generate a distinct phenotype. Here, human elastin-like polypeptide-based ECM-mimic biopolymer was designed by incorporating various cell-adhesion ligands, such as RGD and YIGSR. The significant effects of bioactive fusion ELPs named R-ELP, Y-ELP, and RY-ELP were analyzed for human bone-marrow-derived stem cell adhesion, proliferation, maintenance of stemness properties, and differentiation. Multivalent presentation of variable cell-adhesive ligands on RY-ELP polymers indeed promote efficient cell attachment and proliferation of human fibroblast cells dose-dependently. Similarly, surface modified with RY-ELP promoted strong mesenchymal stem cell (MSCs) attachment with greater focal adhesion (FA) complex formation at 6 h post-incubation. The rate of cell proliferation, migration, population doubling time, and collagen I deposition were significantly enhanced in the presence of RY-ELP compared with other fusion ELPs. Together, the expression of multipotent markers and differentiation capacity of MSCs remained unaffected, clearly demonstrating that stemness properties of MSCs were well preserved when cultured on a RY-ELP-modified surface. Hence, bioactive RY-ELP offers an anchorage support system and effectively induces stimulatory response to support stem cell proliferation. [ABSTRACT FROM AUTHOR]

Rare earth elements (REEs) have become increasingly important materials owing to their use in the high‐tech and clean‐energy industries. However, the unpredictable supply, possible health risks, and environmentally unsustainable extraction practices associated with REEs have encouraged the development of green technologies for the selective extraction and recovery of metals. This study presents a simple and innovative approach for the selective extraction and recovery of total REEs. Elastin‐like polypeptide (ELP) and the REE‐binding domain (lanmodulin) are fused to form REEs‐sensitive and thermo‐responsive genetically encoded ELP called RELP, where ELP offered a reversible, inverse phase transition for repeated uses. The RELP are purified and used for the selective extraction of total REEs from competing non‐REEs metals by controlling the solution temperature (4 and 37 °C) and pH. RELP exhibit high REE specificity, even in the presence of non‐REE metal ions. The bound REEs are readily recovered during at least six repeated cycles, and the efficiency is maintained. Moreover, REEs are selectively recovered by RELP from steel slag leachate, a potential industrial source of REEs. RELP offers a rapid, selective, and scalable method for REE extraction and recovery. This technology can be adapted to recover other precious metals and commodities. [ABSTRACT FROM AUTHOR]

Duck viral hepatitis type I (DVH I) is a lethal disease in ducklings caused by duck hepatitis A virus (DHAV). Although the commercial vaccine is available for vaccination of one-day-old ducklings or breeder ducks, the disease is still prevalent due to the delayed immune response in ducklings and variable maternal antibody levels in breeder duck flocks. To explore the feasibility of duck interferon-α (DuIFN-α) for control of DVH I, DuIFN-α was expressed as an elastin-like polypeptide (ELP) fusion protein (ELP-DuIFN-α) in E. coli and purified by inverse phase transition cycling (ITC). After detection of its cytotoxicity, bioactivity, plasma stability and serum half-life, the protective efficacy of ELP-DuIFN-α against DHAV-1 infection of embryos or ducklings was evaluated using different treatment routes at different infection times. The results show that ELP-DuIFN-α was correctly expressed and purified to more than 90% purity after two cycles of ITC. The purified fusion protein had a specific anti-DHAV-1 activity of 6.0 × 104 IU/mg protein, significantly extended plasma stability and serum half-life without overt cytotoxicity. After allantoic injection with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 5/5, 5/5 or 4/5 embryos survived from the virus challenge. After intramuscular injection or oral administration with ELP-DuIFN-α, 3/5 or 4/5 ducklings survived from co-infection with DHAV-1. After oral administration with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 3/5, 4/5 or 4/5 ducklings survived from the virus challenge, and the relative transcription levels of interferon-stimulated genes were significantly higher than the normal control group and virus challenge control group (p < 0.01). These experimental data suggest that ELP-DuIFN-α can be used as a long-lasting anti-DHAV-1 reagent. [ABSTRACT FROM AUTHOR]

Protein‐based adhesives with their robust adhesion performance and excellent biocompatibility have been extensively explored over years. In particular, the unique adhesion behaviours of mussel and sandcastle worm inspired the development of synthetic adhesives. However, the chemical synthesized adhesives often demonstrate weak underwater adhesion performance and poor biocompatibility/biodegradability, limiting their further biomedical applications. In sharp contrast, genetically engineering endows the protein‐based adhesives the ability to maintain underwater adhesion property as well as biocompatibility/biodegradability. Herein, we outline recent advances in the design and development of protein‐based adhesives by genetic engineering. We summarize the fabrication and adhesion performance of elastin‐like polypeptide‐based adhesives, followed by mussel foot protein (mfp) based adhesives and other sources protein‐based adhesives, such as, spider silk spidroin and suckerin. In addition, the biomedical applications of these bioengineered protein‐based adhesives are presented. Finally, we give a brief summary and perspective on the future development of bioengineered protein‐based adhesives. [ABSTRACT FROM AUTHOR]

Elastin-like polypeptides (ELPs) are bioengineered proteins that have a unique physical property, a thermally triggered inverse phase transition, that can be exploited for drug delivery. ELP-fusion proteins can be used as soluble biologics, thermally targeted drug carriers, self-assembling nanoparticles, and slow-release drug depots. Because of their unique physical characteristics and versatility for delivery of nearly any type of therapeutic, ELP-based drug delivery systems represent a promising platform for biologics development. [ABSTRACT FROM AUTHOR]

Soybean seeds are an ideal host for the production of recombinant proteins because of their high content of proteins, long-term stability of seed proteins under ambient conditions, and easy establishment of efficient purification protocols. In this study, a polypeptide fusion strategy was applied to explore the capacity of elastin-like polypeptide (ELP) and γ-zein fusions in increasing the accumulation of the recombinant protein in soybean seeds. Transgenic soybean plants were generated to express the γ-zein- or ELP-fused green fluorescent protein (GFP) under the control of the soybean seed-specific promoter of β-conglycinin alpha subunit (BCSP). Significant differences were observed in the accumulation of zein-GFP and GFP-ELP from that of the unfused GFP in transgenic soybean seeds based on the total soluble protein (TSP), despite the low-copy of T-DNA insertions and similar expression at the mRNA levels in selected transgenic lines. The average levels of zein-GFP and GFP-ELP accumulated in immature seeds of these transgenic lines were 0.99% and 0.29% TSP, respectively, compared with 0.07% TSP of the unfused GFP. In mature soybean seeds, the accumulation of zein-GFP and GFP-ELP proteins was 1.8% and 0.84% TSP, an increase of 3.91- and 1.82-fold, respectively, in comparison with that of the unfused GFP (0.46% TSP). Confocal laser scanning analysis showed that both zein-GFP and GFP-ELP were abundantly deposited in many small spherical particles of transgenic seeds, while there were fewer such florescence signals in the same cellular compartments of the unfused GFP-expressing seeds. Despite increased recombinant protein accumulation, there were no significant changes in the total protein and oil content in seeds between the transgenic and non-transformed plants, suggesting the possible presence of threshold limits of total protein accumulation in transgenic soybean seeds. Overall, our results indicate that γ-zein and ELP fusions significantly increased the accumulation of the recombinant protein, but exhibited no significant influence on the total protein and oil content in soybean seeds. [ABSTRACT FROM AUTHOR]

Patients with chronic kidney disease (CKD) have a high cardiovascular mortality. CKD and heart failure (HF) coexist in up to 50% of patients, and both associate with inflammation. We aimed to define the cardiac phenotype of a novel swine model of CKD and test the hypothesis that inflammation of renal origin propels the development of precursors of HF in CKD. CKD was induced in 14 pigs, which were followed for 14 wk. Renal (multidetector computed tomography) and cardiac (echocardiography) hemodynamics were quantified before and 8 wk after single intrarenal administration of placebo or a biopolymer-fused peptide inhibitor of NF-κB that blocks NF-κB activity and decreases inflammatory activity (SynB1-ELP-p50i). Blood was collected to quantify cytokines (TNF-α, monocyte chemoattractant protein-1, and interleukins), markers of inflammation (C-reactive protein), and biomarkers of HF (atrial and brain natriuretic peptides). Pigs were then euthanized, and kidneys and hearts were studied ex vivo. Normal pigs were used as time-matched controls. Renal dysfunction in CKD was accompanied by cardiac hypertrophy and fibrosis, diastolic dysfunction, increased renal and cardiac expression of TNF-α, monocyte chemoattractant protein-1, and interleukins, canonical and noncanonical mediators of NF-κB signaling, circulating inflammatory factors, and biomarkers of HF. Notably, most of these changes were improved after intrarenal SynB1-SynB1-ELP-p50i, although cardiac inflammatory signaling remained unaltered. The translational traits of this model support its use as a platform to test novel technologies to protect the kidney and heart in CKD. A targeted inhibition of renal NF-κB signaling improves renal and cardiac function, suggesting an inflammatory renal-cardio axis underlying early HF pathophysiology in CKD. [ABSTRACT FROM AUTHOR]

Elastin-like polypeptides (ELP) have been used as a genetically-engineered, biocompatible substitute for elastin. Cell culture coatings prepared using ELP conjugated to low molecular weight polyethyleneimine (PEI) entices cells to form three-dimensional cellular aggregates that mimic their in vivo counterparts. This study seeks to control the deposition of the ELP and ELP-PEI molecules to control the roughness of the final coatings. The two polymers were coated onto three different substrates (glass, polystyrene, tissue-culture polystyrene) and the solution environment was altered by changing the polymer concentration (0.5, 1.0, 1.5 mg/mL) and/or salt concentration (None, 0.2 M phosphate buffered saline) for a total of 36 conditions. Atomic force microscopy (AFM) was used to measure the average roughness (Ra) of the samples and found that ELP coated samples had a higher Ra than their ELP-PEI counterparts. The coatings were tested for stability by performing cell culture media changes every three days for 11 days. AFM showed that the average roughness of the tested samples increased with each media change. To address this, the surfaces were crosslinked using hexamethyl diisocyanate, which minimized the change in surface roughness even when subjected to an intense sonication process. This study provides parameters to achieve elastin-based coatings with controlled roughness that can be used to support stable, long-term in vitro cell culture. [ABSTRACT FROM AUTHOR]

The extracellular matrix components laminin and elastin serve key roles in stem cell therapy. Elastin-like polypeptides (ELPs), derived from a soluble form of elastin, affect the proliferation and differentiation of various types of cells. In the present study, a novel protein was designed containing globular domains 1-3 of laminin α5 (Lα5LG1-3) fused to ELPs (Lα5LG1-3/ELP). Lα5LG1-3/ELP was expressed in Escherichia coli and displayed a molecular size of ~70 kDa on 12% SDS-polyacrylamide gels. The cellular activities, such as cellular adhesion (adhesion assay) and proliferation (MTT cytotoxicity assay), of human mesenchymal stem cells (hMSCs) treated with 1 μg/ml of Lα5LG1-3/ELP were enhanced compared with those of untreated cells. Additionally, the number of undifferentiated hMSCs and their degree of stemness were assessed based on the gene expression levels of the stem cell markers cluster differentiation 90 (CD90), endoglin (CD105) and CD73. The expression levels of these markers were upregulated by 2.42-, 2.29- and 1.92-fold, respectively, in the hMSCs treated with Lα5LG1-3/ELP compared with the levels in untreated controls. Thus, Lα5LG1-3/ELP may be used to enhance the viability of hMSCs and preserve their undifferentiated state, whereby the clinical applications of hMSCs may be improved. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of the incorporation of elastin‐like polypeptide (ELP) on the adhesion maturation of mineral trioxide aggregates (MTA). Two types of ELPs (V125 and V125E8) were genetically synthesized. V125 consisted of 125 repeating pentapeptides (Val‐Pro‐Gly‐Xaa‐Gly) and V125E8 was functionalized with octaglutamic acid in the C‐terminus of V125; both were diluted to 10 wt% in solution. Three 1.5 mm diameter holes in dentin discs were filled with MTA mixed with either a solution of ELP or deionized water. Push‐out bond strength tests were performed following storage in simulated body fluid (SBF) for 1, 2, 4, and 8 weeks (n = 12). The interface between dentin and MTA was observed via scanning electron microscopy (SEM). The maturation of MTA was evaluated with a stereoscopic microscope and SEM. The incorporation of a specific ELP (V125E8) significantly increased the bond strength of MTA to dentin with regard to every maturation period (p < 0.05). The bond strength of MTA also significantly increased with a longer maturation time irrespective of ELP incorporation (p < 0.05). V125E8‐incorporated MTA exhibited a more intimate interface with dentin compared to the other groups. More spindle‐shaped crystal structures and thicker crystals were observed in all MTA mixtures as the storage duration increased even though V125E8 exhibited fewer crystal structures on the surface. Within the limitations of this study, the incorporation of V125E8 increased the adhesive properties of MTA and the maturation of MTA occurred regardless of ELP incorporation. [ABSTRACT FROM AUTHOR]

While three‐dimensional spheroids outperform traditional two‐dimensional monolayer culture for human adipose‐derived stem cells (hASCs), there is not a consensus on the most successful method for enhancing their adipogenic differentiation and minimizing the loss of physiologically relevant, fatty spheroids during culture. To this end, we compared three culture methods, namely, elastin‐like polypeptide‐polyethyleneimine (ELP‐PEI) coated surfaces, ultra‐low attachment static culture, and suspension culture for their ability to form and retain productive hASC spheroids. The ELP‐PEI coatings used the ELP conjugated to two molecular weights of PEI (800 or 25,000 g/mol). FTIR spectroscopy, atomic force microscopy, and contact angle goniometry revealed that the ELP‐PEI coatings had similar chemical structures, surface topography, and hydrophobicity. Time‐lapse microscopy showed that increasing the PEI molecular weight resulted in smaller spheroids. Measurement of triglyceride content showed that the three methods induced comparable differentiation of hASCs toward the adipogenic lineage. DNA content and morphometric analysis revealed merging of spheroids to form larger spheroids in the ultra‐low attachment static culture and suspension culture methods. In contrast, the retention of hASC spheroid sizes and numbers with a regular spheroid size (~100 μm) were best atop the ELP‐PEI800 coatings. Overall, this research shows that the spheroid culture atop the ELP‐PEI coatings is a suitable cell culture model for future studies involving long‐term, three‐dimensional culture of mature adipocytes derived from hASCs. [ABSTRACT FROM AUTHOR]

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Herein, we presented a novel strategy for the preparation of peptide/SiO2 nanoparticles enabling enzyme covalent immobilization by combining elastin-like peptide (ELP)-based biomimetic silicification for SiO2 synthesis with the SpyCatcher/SpyTag system for spontaneous covalent bond formation. Using this method, the nanocarriers (SpyCatcher-ELP@SiO2) can be prepared in a one-step process within minutes under mild conditions without additional surface modification. These nanocarriers could directly immobilize the SpyTag-fused target enzymes from the crude cell lysate without prepurification. With the nanocarriers, two enzymes (xylanase and lichenase) used in biomass conversion were successfully immobilized with high activity recovery (>90%) and good reusability (>80%). [ABSTRACT FROM AUTHOR]

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and cost-effective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic. [ABSTRACT FROM AUTHOR]

Background and Aim recombinant DNA technique is a powerful and appropriate method for the production of protein biopolymers with specificity in amino acid sequence and spatial chemistry. Elastin-Like Polypeptide (ELP) is a biocompatible, biodegradable and non-immunological biopolymer used in various biotechnology studies. The ELP tag is a cheap, fast and non-chromatographic technique for purifying target proteins. In this study, pET expression vector was designed for the combination of ELP gene sequences and target recombinant protein in order to produce recombinant fusion protein with the ELP tag. Methods & Materials MOD gene was transformed to E. coli-BL21 (DE3) cells after designing and synthesis among the XbaI and XhoI restriction sites in the pET-32a (+) vector of the clone. Then, colonies were isolated based on plasmid size and examined by cutting using restriction enzymes. The final recombinant colonies was verified using polymerase chain reaction method and DNA sequencing. Ethical Considerations The Research Ethics Committee of Arak University of Medical Sciences approved all ethical considerations ofworking on laboratory animals (Code: 92-146-11). Results Replacing the MOD sequence in the pET-32a vector (+) eliminated the components expressing the fusion tags (Thioredoxin, Histidine, and S-tag), the identification site of protease enzyme (tobacco etch virus), and multiple cloning site. In addition, it added specific restriction enzyme identification sequences of ELP gene and target gene. As a result, in the optimized pET-MODvector, 466 nucleotides reduced in size and the secondary structure was improved. Conclusion Considering the improvement of spatial structure and reduction of pET-MOD vector size, as well as the possibility of the fusion of recombinant protein with the ELP tag, it is possible to use this vector for ELPyation of the target protein. [ABSTRACT FROM AUTHOR]

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification. [ABSTRACT FROM AUTHOR]

Abstract: Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters. [ABSTRACT FROM AUTHOR]

Musculoskeletal infections caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa in children and adults can lead to adverse outcomes including a need for extensive surgical debridement and limb amputation. To enable targeted antimicrobial release in infected tissues, the objective of this study was to design and investigate novel elastin-like polypeptide (ELP)-based thermally sensitive liposomes in vitro. ELP biopolymers can change their phase behaviour at higher temperatures. We hypothesised that ELP-TSL will improve therapeutic efficacy by releasing antimicrobial payloads locally at higher temperatures (≥39 °C). ELP-TSL library were formulated by varying cholesterol and phospholipid composition by the thin film and extrusion method. A broad-spectrum antimicrobial (Ciprofloxacin or Cipro) was encapsulated inside the liposomes by the ammonium sulphate gradient method. Cipro release from ELP-TSLs was assessed in physiological buffers containing ∼25% serum by fluorescence spectroscopy, and efficacy against Staphylococcus aureus and Pseudomonas aeruginosa was assessed by disc diffusion and planktonic assay. Active loading of Cipro achieved an encapsulation efficiency of 40-70% in the ELP-TSL depending upon composition. ELP-TSL Cipro release was near complete at ≥39 °C; however, the release rates could be delayed by cholesterol. Triggered release of Cipro from ELP-TSL at ∼42 °C induced significant killing of S. aureus and P. aeruginosa compared to 37 °C. Our in vitro data suggest that ELP-TSL may potentially improve bacterial wound therapy in patients. [ABSTRACT FROM AUTHOR]

Objective: To design a fusion polypeptide combining functions of self-assembly and purification for immobilizing enzymes.Results: A collagen-like polypeptide (CLP) was fused to an elastin-like polypeptide (ELP) through genetic engineering. CLP-ELP was separately fused to superoxide dismutase (SOD) and D-amino acid oxidase (DAAO). The recombinant enzymes were purified with using reversible phase transition. The interfering effect of H2O2 on the secondary structures of the recombinant enzymes was significantly reduced. The stability of the recombinant enzymes against denaturing by urea was improved. SOD-CLP-ELP exhibited a proteinaceous microporous network, and DAAO-CLP-ELP exhibited micro-clusters. The superoxide anion (•O2−) scavenging ability of SOD-CLP-ELP was 1.5 times that of SOD, and the catalytic efficiency of DAAO-CLP-ELP was 1.7 times that of DAAO.Conclusions: The advantages of the CLP-ELP-fused enzymes have been demonstrated and CLP-ELP can be used to immobilize other enzymes/proteins. [ABSTRACT FROM AUTHOR]

We have recently developed a novel double-hydrophobic elastin-like triblock polypeptide called GPG, designed after the uneven distribution of two different hydrophobic domains found in elastin, an extracellular matrix protein providing elasticity and resilience to tissues. Upon temperature trigger, GPG undergoes a sequential self-assembling process to form flexible beaded nanofibers with high homogeneity and excellent dispersibility in water. Given that GPG might be a potential elastin-mimetic material, we sought to explore the biological activities of this block polypeptide. Besides GPG, several functionalized derivatives were also constructed by fusing functional motifs such as KAAK or KAAKGRGDS at the C-terminal of GPG. Although the added motifs affected the kinetics of fiber formation and β-sheet contents, all three GPGs assembled into beaded nanofibers at the physiological temperature. The resulting GPG nanofibers preserved their beaded structures in cell culture medium; therefore, they were coated on polystyrene substrates to study their cytocompatibility toward mouse embryonic fibroblasts, NIH-3T3. Among the three polypeptides, GPG having the cell-binding motif GRGDS derived from fibronectin showed excellent cell adhesion and cell proliferation properties compared to other conventional materials, suggesting its promising applications as extracellular matrices for mammalian cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2475-2484, 2017. [ABSTRACT FROM AUTHOR]

Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood–brain barrier penetration. The SynB1–ELP1–dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes’ expression. SynB1–ELP1–dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1–ELP1–dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition. [ABSTRACT FROM PUBLISHER]

Herein, we demonstrate a new approach for small-caliber vascular reconstruction using a non-porous elastin-like polypeptide hydrogel tubular scaffold, based on the concept of guided vascular regeneration (GVR). The scaffolds are composed of elastin-like polypeptide, (Val-Pro-Gly-Ile-Gly) n, for compliance matching and antithrombogenicity and an Arg-Gly-Asp (RGD) motif for connective tissue regeneration. When the polypeptide was mixed with an aqueous solution of β-[Tris(hydroxymethyl)phosphino]propionic acid at 37°C, the polypeptide hydrogel was rapidly formed. The elastic modulus of the hydrogel was 4.4 kPa. The hydrogel tubular scaffold was formed in a mold and reinforced with poly(lactic acid) nanofibers. When tubular scaffolds with an inner diameter of 1 mm and length of 5 mm were implanted into rat abdominal aortae, connective tissue grew along the scaffold luminal surface from the flanking native tissues, resulting in new blood vessel tissue with a thickness of 200 μm in 1 month. In contrast, rats implanted with control scaffolds without the RGD motif died. These results indicate that the non-porous hydrogel tubular scaffold containing the RGD motif effectively induced rapid tissue regeneration and that GVR is a promising strategy for the regeneration of small-diameter blood vessels. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1746-1755, 2017. [ABSTRACT FROM AUTHOR]

3D culture systems have the ability to mimic the natural microenvironment by allowing better cell-cell interactions. We have prepared an in vitro 3D osteogenic cell culture model using human adipose derived stem cells (hASCs) cultured atop recombinant elastin-like polypeptide (ELP) conjugated to a charged polyelectrolyte, polyethyleneimine (PEI). We demonstrate that hASCs cultured atop the ELP-PEI coated tissue culture polystyrene (TCPS) formed 3D spheroids and exhibited superior differentiation toward osteogenic lineage compared to the traditional two dimensional (2D) monolayer formed atop uncoated TCPS. Live/dead viability assay confirmed >90% live cells at the end of the 3-week culture period. Over the same culture period, higher protein content was observed in 2D monolayer than 3D spheroids, as the 2D environment allowed continued proliferation, while 3D spheroids underwent contact-inhibited growth arrest. The normalized alkaline phosphatase (ALP) activity, which is an indicator for early osteogenic differentiation was higher for 3D spheroids. The normalized osteocalcin (OCN) production, which is an indicator for osteogenic maturation was also higher for 3D spheroids while 2D monolayer had no noticeable OCN production. On day 22, increased Alizarin red uptake by 3D spheroids showed greater mineralization activity than 2D monolayer. Taken together, these results indicate a superior osteogenic differentiation of hASCs in 3D spheroid culture atop ELP-PEI coated TCPS surfaces than the 2D monolayer formed on uncoated TCPS surfaces. Such enhanced osteogenesis in 3D spheroid stem cell culture may serve as an alternative to 2D culture by providing a better microenvironment for the enhanced cellular functions and interactions in bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1230-1236, 2017. [ABSTRACT FROM AUTHOR]

Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastinlike polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH2-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with salinetreated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies. [ABSTRACT FROM AUTHOR]

A heme-dependent peroxidase (HDP) catalyzes the ortho-hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) in the presence of hydrogen peroxide. L-DOPA can be used for the treatment of Parkinson's disease. In this work, to improve the catalytic efficiency, the heme-dependent peroxidase has been genetically modified with an elastin-like polypeptide (ELP). bicinchoninic acid (BCA) assay demonstrated that HDP-ELP has a higher solubility in aqueous solutions than HDP. Circular dichroism (CD) spectra showed that HDP-ELP has a higher stability than HDP. Enzyme kinetics has been investigated over a range of substrate concentrations. It has been demonstrated that HDP-ELP exhibited a catalytic efficiency 2.4 times that of HDP. [ABSTRACT FROM AUTHOR]

Background: Brachytherapy is a targeted type of radiotherapy utilized in the treatment of cancers. Elastin-like polypeptides are a unique class of genetically engineered peptide polymers that have several attractive properties for brachytherapy.Aims: To explore the feasibility and application of brachytherapy for VX2 liver tumor using elastin-like polypeptides with (131)I so as to provide reliable experimental evidence for a new promising treatment of liver cancer.Methods: Elastin-like polypeptide as carrier was labeled with (131)I using the iodogen method. Ten eligible rabbits with VX2 liver tumor were randomly divided into the treatment group (n = 5) and control group (n = 5). The treatment group received brachytherapy using elastin-like polypeptide with (131)I, and in the control group, elastin-like polypeptide was injected into the VX2 liver tumor as a control. Periodic biochemical and imaging surveillances were required to assess treatment efficacy.Results: The stability of elastin-like polypeptide with (131)I in vitro was maintained at over 96.8 % for 96 h. Biochemistry and imaging indicated brachytherapy using elastin-like polypeptide with (131)I for liver tumor can improve liver function and inhibit tumor growth (P < 0.05).Conclusions: Elastin-like polypeptide can be an ideal carrier of (131)I and have high labeling efficiency, radiochemical purity and stability. Brachytherapy using elastin-like polypeptide with (131)I for liver tumor is a useful therapy that possesses high antitumor efficacy advantages. [ABSTRACT FROM AUTHOR]

D-amino acid oxidase (DAAO) and catalase (CAT) have been genetically modified by fusing them to an elastin-like polypeptide (ELP). ELP-DAAO and ELP-CAT have been separately immobilized on multi-walled carbon nanotubes (MWNTs). It has been found that the secondary structures of the enzymes have been preserved. ELP-DAAO catalyzed the oxidative deamination of D-alanine, and H2O2 was evolved continuously. When the MWNT-supported enzymes were used together, the generated hydrogen peroxide of ELP-DAAO could be decomposed in situ. The catalytic efficiency of the two immobilized enzymes was more than five times greater than that of free ELP-DAAO when the ratio of immobilized ELP-CAT to immobilized ELP-DAAO was larger than 1:1. [ABSTRACT FROM AUTHOR]

Objectives: An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase. Results: R- ω-Transaminase ( R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP- R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, ( R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP- R- ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %. Conclusions: The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports. [ABSTRACT FROM AUTHOR]

Elastin-like polypeptides (ELPs) are a class of stimuli-responsive biopolymers inspired by the intrinsically disordered domains of tropoelastin that are composed of repeats of the VPGXG pentapeptide motif, where X is a “guest residue”. They undergo a reversible, thermally triggered lower critical solution temperature (LCST) phase transition, which has been utilized for a variety of applications including protein purification, affinity capture, immunoassays, and drug delivery. ELPs have been extensively studied as protein polymers and as biomaterials, but their relationship to other disordered proteins has heretofore not been established. The biophysical properties of ELPs that lend them their unique material behavior are similar to the properties of many intrinsically disordered proteins (IDP). Their low sequence complexity, phase behavior, and elastic properties make them an interesting “minimal” artificial IDP, and the study of ELPs can hence provide insights into the behavior of other more complex IDPs. Motivated by this emerging realization of the similarities between ELPs and IDPs, this review discusses the biophysical properties of ELPs, their biomedical utility, and their relationship to other disordered polypeptide sequences. [ABSTRACT FROM AUTHOR]

The phase-transition temperatures of an elastin-like polypeptide (ELP) with the (GVGVP)40 sequence and solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity in its aqueous solutions were quantified in the absence and presence of different salts (Na2SO4, NaCl, NaClO4, and NaSCN) and various osmolytes (sucrose, sorbitol, trehalose, and trimethylamine N-oxide (TMAO)). All osmolytes decreased the ELP phase-transition temperature, whereas NaCl and Na2SO4 decreased, and NaSCN and NaClO4 increased it. The determined phase-transition temperatures may be described as a linear combination of the solvent's dipolarity/polarizability and hydrogen-bond donor acidity. The linear relationship established for the phase-transition temperature in the presence of salts differs quantitatively from that in the presence of osmolytes, in agreement with different (direct and indirect) mechanisms of the influence of salts and osmolytes on the ELP phase-transition temperature. [ABSTRACT FROM AUTHOR]

ABSTRACT Preparation of hydrogels that possess an effective antibiotic release profile and better mechanical properties compared to the traditionally used collagen hydrogels has the potential to minimize post-surgical infections and support wound healing. Toward this goal, we prepared elastin-like polypeptide (ELP)-collagen composite hydrogels that displayed a significantly higher elastic modulus compared to the collagen hydrogels. We then characterized the release behavior of the collagen and ELP-collagen hydrogels loaded with varying dosages (1-5% w/w) of a commonly used broad spectrum antibiotic, doxycycline hyclate. Both collagen and ELP-collagen hydrogels showed a gradual time dependent doxycycline release over a period of 5 days. The ELP-collagen hydrogels, in general, showed a slower release of the doxycycline compared to the collagen hydrogels. The released doxycycline was found to be effective against four bacterial strains ( Escherichia coli, Pseudomonas aeruginosa, Streptococcus sanguinis, and methicillin-resistant Staphylococcus aureus) in a dose dependent manner. Combined with their improved mechanical properties, the gradual and effective drug release from the biocompatible ELP-collagen hydrogels shown here may be beneficial for drug delivery and tissue engineering applications. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 782-790, 2015. [ABSTRACT FROM AUTHOR]

Background: Pregnant females are largely overlooked in drug development due to concerns for fetal health. Additionally, pregnancy is often an exclusion criterion in clinical trials, so the safety of many drugs during pregnancy is unknown. Purpose: The goal of this study was to evaluate Elastin-like Polypeptide (ELP), a synthetic protein derived from human elastin, for maternally sequestered drug delivery. ELP is a versatile drug carrier with a long plasma half-life, low immunogenicity, and the ability to be fused to nearly any small molecule or protein-based therapeutic. Methods: We determined the pharmacokinetics, biodistribution, and fetal exposure to the ELP drug carrier using quantitative fluorescence techniques in a rat pregnancy model. Results: After either bolus IV administration or continuous infusion over five days, ELPs accumulated strongly in the kidneys, liver, and placenta, but importantly, little to no ELPs were detectable in the fetus. Within the placenta, ELPs were localized to the chorionic plate and broadly distributed within the labyrinth, but were excluded from the fetal portion of the chorionic villi. Conclusion: These data indicate that ELP does not cross the placenta, and they suggest that this adaptable drug delivery system is a promising platform for prevention of fetal drug exposure. [ABSTRACT FROM AUTHOR]

Purpose: Hepatocellular carcinoma (HCC) suffers high tumour recurrence rate after thermal ablation. Heat shock protein 90 (Hsp90) induced post-ablation is critical for tumour survival and progression. A combination therapy of thermal ablation and polymer conjugated Hsp90 chemotherapy was designed and evaluated for complete tumour eradication of HCC. Materials and methods: A thermo-responsive, elastin-like polypeptide (ELP)-based tri-block biopolymer was developed and conjugated with a potent Hsp90 inhibitor, geldanamycin (GA). The anti-cancer efficacy of conjugates was evaluated in HCC cell cultures with and without hyperthermia (43 °C). The conjugates were also administered twice weekly in a murine HCC model as a single treatment or in combination with single electrocautery as the ablation method. Results: ELP-GA conjugates displayed enhanced cytotoxicity in vitro and effective heat shock inhibition under hyperthermia. The conjugates alone significantly slowed the tumour growth without systemic toxicity. Four doses of thermo-responsive ELP-GA conjugates with concomitant simple electrocautery accomplished significant Hsp90 inhibition and sustained tumour suppression. Conclusion: Hsp90 inhibition plays a key role in preventing the recurrence of HCC, and the combination of ablation with targeted therapy holds great potential to improve prognosis and survival of HCC patients. [ABSTRACT FROM AUTHOR]

The development of protein-based carriers for drug delivery has been well studied. We previously constructed a protein-based nanoparticle consisting of genetically engineered elastin-like polypeptides (ELPs) with a fused poly-aspartic acid tail (ELPD). The size of the self-assembled ELPD nanoparticles was regulated by charged repulsion of the poly-aspartic acid chains. In the present study, epidermal growth factor (EGF) was genetically fused to the C-terminus of ELPD to impart an active targeting ability to the ELPD nanoparticles. We examined the nanoparticle formation with EGF as well as its targeting ability. ELPD with fused EGF was found to form nanoparticles that displayed multivalent EGFs on their surface. EGF-displayed nanoparticles loaded with the anti-cancer drug paclitaxel were internalized into cells overexpressing the EGF receptor, and induced cell death. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1792-1798, 2014. [ABSTRACT FROM AUTHOR]