Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and ...efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP5 to ELP50 was constructed using recursive directional ligation by plasmid reconstruction. ELP50 was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP50, which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP50 expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP50 was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP50 purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP50 had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP50 could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.
•Construct a small gene library of ELP from ELP5 to ELP50.•Purify and produce the fusion EG12B-ELP50 by inverse transition cycling.•Improve the thermostability of EG12B by fusion with ELP50.•Provide a rapid and simple strategy for target protein purification.
Elastin is an important protein of the extracellular matrix of higher vertebrates, which confers elasticity and resilience to various tissues and organs including lungs, skin, large blood vessels and ...ligaments. Owing to its unique structure, extensive cross-linking and durability, it does not undergo significant turnover in healthy tissues and has a half-life of more than 70 years. Elastin is not only a structural protein, influencing the architecture and biomechanical properties of the extracellular matrix, but also plays a vital role in various physiological processes. Bioactive elastin peptides termed elastokines - in particular those of the GXXPG motif - occur as a result of proteolytic degradation of elastin and its non-cross-linked precursor tropoelastin and display several biological activities. For instance, they promote angiogenesis or stimulate cell adhesion, chemotaxis, proliferation, protease activation and apoptosis. Elastin-degrading enzymes such as matrix metalloproteinases, serine proteases and cysteine proteases slowly damage elastin over the lifetime of an organism. The destruction of elastin and the biological processes triggered by elastokines favor the development and progression of various pathological conditions including emphysema, chronic obstructive pulmonary disease, atherosclerosis, metabolic syndrome and cancer. This review gives an overview on types of human elastases and their action on human elastin, including the formation, structure and biological activities of elastokines and their role in common biological processes and severe pathological conditions.
Blood vessels are dynamic structures composed of cells and extracellular matrix (ECM), which are in continuous cross-talk with each other. Thus, cellular changes in phenotype or in ...proliferation/death rate affect ECM synthesis. In turn, ECM elements not only provide the structural framework for vascular cells, but they also modulate cellular function through specific receptors. These ECM-cell interactions, together with neurotransmitters, hormones and the mechanical forces imposed by the heart, modulate the structural organization of the vascular wall. It is not surprising that pathological states related to alterations in the nervous, humoral or haemodynamic environment-such as hypertension-are associated with vascular wall remodeling, which, in the end, is deleterious for cardiovascular function. However, the question remains whether these structural alterations are simply a consequence of the disease or if there are early cellular or ECM alterations-determined either genetically or by environmental factors-that can predispose to vascular remodeling independent of hypertension. Elastic fibres might be key elements in the pathophysiology of hypertensive vascular remodeling. In addition to the well known effects of hypertension on elastic fibre fatigue and accelerated degradation, leading to loss of arterial wall resilience, recent investigations have highlighted new roles for individual components of elastic fibres and their degradation products. These elements can act as signal transducers and regulate cellular proliferation, migration, phenotype, and ECM degradation. In this paper, we review current knowledge regarding components of elastic fibres and discuss their possible pathomechanistic associations with vascular structural abnormalities and with hypertension development or progression.
Elastin‐like peptides (ELPs) are a versatile platform for tissue engineering and drug delivery. Here, micelle forming ELP chains are genetically fused to three therapeutic molecules, keratinocyte ...growth factor (KGF), stromal cell‐derived growth factor 1 (SDF1), and cathelicidin (LL37), to be used in wound healing. Chronic wounds represent a growing problem worldwide. A combinatorial therapy approach targeting different aspects of wound healing would be beneficial, providing a controlled and sustained release of active molecules, while simultaneously protecting these therapeutics from the surrounding harsh wound environment. The results of this study demonstrate that the conjugation of the growth factors KGF and SDF1 and the antimicrobial peptide LL37 to ELPs does not affect the micelle structure and that all three therapeutic moieties retain their bioactivity in vitro. Importantly, when the combination of these micelle ELP nanoparticles are applied to wounds in diabetic mice, over 90 % wound closure is observed, which is significantly higher than when the therapeutics are applied in their naked forms. The application of the nanoparticles designed here is the first report of targeting different aspect of wound healing synergistically.
Micelle‐forming elastin‐like peptides (ELPs) are genetically fused to therapeutic proteins (keratinocyte growth factor, stromal cell derived factor 1, and antimicrobial peptide cathelicidin). The fusion proteins performed better than the naked, recombinant therapeutics (as well as the phosphate‐buffered saline control and empty ELP particles), in a diabetic wound healing mouse model, demonstrating the advantage of the micelle ELP platform for therapeutics delivery.
In this work, an aggregation-induced emission (AIE) molecule (tetraphenylethene derivative, TPE-COOH) was conjugated to elastin-like polypeptides (ELPs40) via an amide bond to form ELPs40-TPE. The ...successful synthesis of ELPs40-TPE was confirmed by Circular Dichroism spectroscopy, gel electrophoresis, UV-vis absorption, and fluorescence emission spectroscopy. ELPs40-TPE possessed both amphiphilicity and the features of an AIE, and the fluorescence intensity was dependent on the local temperature. The Hela cells imaging indicated that ELPs40-TPE has great potential for bio-imaging applications because of its advantages of high fluorescence intensity, good water-solubility, and remarkable biocompatibility.
This study aimed to investigate the protective effects of bovine elastin hydrolysates on UV-induced skin photoaging in mice and to identify the potent antiphotoaging peptides. Results showed that the ...ingestion of elastin peptides could obviously ameliorate epidermis hyperplasia and fibroblast apoptosis, and increase the content of hydroxyproline and water in photoaging skin in vivo (p < 0.05). Furthermore, four peptides with elastase inhibitory activity were purified and identified, including GLPY, PY, GLGPGVG, and GPGGVGAL. Interestingly, GLPY and GPGGVGAL exhibited the highest inhibition activity with 58.77% and 42.91% at 10 mΜ, respectively. This might be attributed to the N-terminal Gly, C-terminal Leu, and Pro at the third position of the N-terminus, which showed stronger affinity and interaction with elastase. Moreover, GLPY and GPGGVGAL could also inhibit the apoptosis of fibroblasts effectively at 50 μΜ (p < 0.01). It suggested that elastin peptides had great potential to prevent and regulate skin photoaging.
Fibrosis is the common endpoint and currently the best predictor of progression of chronic kidney diseases (CKDs). Despite several drawbacks, biopsies remain the only available means to specifically ...assess the extent of renal fibrosis. Here, we show that molecular imaging of the extracellular matrix protein elastin allows for noninvasive staging and longitudinal monitoring of renal fibrosis. Elastin was hardly expressed in healthy mouse, rat, and human kidneys, whereas it was highly up-regulated in cortical, medullar, and perivascular regions in progressive CKD. Compared to a clinically relevant control contrast agent, the elastin-specific magnetic resonance imaging agent ESMA specifically detected elastin expression in multiple mouse models of renal fibrosis and also in fibrotic human kidneys. Elastin imaging allowed for repetitive and reproducible assessment of renal fibrosis, and it enabled longitudinal monitoring of therapeutic interventions, accurately capturing anti-fibrotic therapy effects. Last, in a model of reversible renal injury, elastin imaging detected ensuing fibrosis not identifiable via routine assessment of kidney function. Elastin imaging thus has the potential to become a noninvasive, specific imaging method to assess renal fibrosis.
The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our ...laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.
Peptide drugs are an exciting class of pharmaceuticals for the treatment of a variety of diseases; however, their short half-life dictates multiple and frequent injections causing undesirable side ...effects. Herein, we describe a novel peptide delivery system that seeks to combine the attractive features of prolonged circulation time with a prolonged release formulation. This system consists of glucagon-like peptide-1, a type-2 diabetes drug fused to a thermally responsive, elastin-like-polypeptide (ELP) that undergoes a soluble–insoluble phase transition between room temperature and body temperature, thereby forming an injectable depot. We synthesized a set of GLP-1-ELP fusions and verified their proteolytic stability and potency in vitro. Significantly, a single injection of depot forming GLP-1-ELP fusions reduced blood glucose levels in mice for up to 5days, 120 times longer than an injection of the native peptide. These findings demonstrate the unique advantages of using ELPs to release peptide-ELP fusions from a depot combined with enhanced systemic circulation to create a tunable peptide delivery system.
A fusion of glucagon-like peptide-1 and elastin-like polypeptide is able to reduce blood glucose for over 5days after a single injection by forming an extended release subcutaneous depot and prolonging peptide circulation time. Display omitted
Chronic obstructive pulmonary disease (COPD) is characterized by high levels of protease activity leading to degradation of elastin followed by loss of elasticity of the lung and the development of ...emphysema. Elastin is an essential structural component of the lung parenchyma to support the expansion and recoil of the alveoli during breathing. The lung extracellular matrix is vulnerable to pathological structural changes upon upregulation of serine proteases, including cathepsin G (CG) and proteinase 3 (PR3). In this study, we explored the diagnostic features of elastin neo-epitopes generated by CG and PR3. Two novel competitive enzyme-linked immunosorbent assays (ELISA) measuring CG and PR3 generated elastin fragments (EL-CG and ELP-3 respectively) were developed for assessment in serum. Both assays were technically robust and biologically validated in serum from patients with COPD. Serological levels of both elastin fragments were significantly elevated in patients with COPD compared to healthy controls. These data suggest that EL-CG and ELP-3 may serve as plausible biologic markers of destructive changes in COPD.
•Identification of elastin fragments specifically generated by cathepsin G (CG) and proteinase 3 (PR3) respectively.•Development of the two assays EL-CG and ELP-3 which are specific for the CG or PR3 degraded elastin neo-epitopes.•Significant higher concentrations of EL-CG and ELP-3 in patients diagnosed with COPD compared to healthy subjects.
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