Elastin-like Proteins Research Articles

Independent tuning of multiple biomaterial properties using protein engineering

A key attribute missing from many current biomaterials is the ability to independently tune multiple biomaterial properties without simultaneously affecting other material parameters. Because cells are well known to respond to changes in the initial elastic modulus, degradation rate, and cell adhesivity of a biomaterial, it is critical to develop synthetic design strategies that allow decoupled tailoring of each individual parameter in order to systematically optimize cell-scaffold interactions. We present the development of a biomimetic scaffold composed of chemically crosslinked, elastin-like proteins designed to support neural regeneration through a combination of cell adhesion and cell-induced degradation and remodeling. The design of these engineered proteins includes cell adhesion sequences to enable neuronal attachment as well as sequences sensitive to cleavage by urokinase plasminogen activator (uPA), a protease locally secreted from the tips of growing neurites, to enable highly localized and tunable degradation properties. These engineered proteins are produced using recombinant techniques and chemically crosslinked into highly swollen hydrogels with controllable mechanical properties. Through a modest 3% change in the chemical identity of three otherwise identical engineered proteins, we can modify the uPA substrate specificity resulting in tunable changes in protease degradation half-life over two orders of magnitude. Under high uPA exposure, the designed scaffolds exhibit systematic variation of scaffold lifetime, from being fully degraded within a single day to showing no noticeable degradation within a full week. In vitro studies using the model PC-12 neuronal-like cell line show that the crosslinked proteins support tunable cell adhesion and neuronal differentiation. Increasing the density of RGD peptides present in the protein substrates leads to increased cell adhesion and more extensive neurite outgrowth. These engineered proteins offer the ability to independently tailor the mechanics, degradation properties, and cell adhesivity of scaffolds for the study of central nervous system regeneration.

Expression of Sox and fibrillar collagen genes in lamprey larval chondrogenesis with implications for the evolution of vertebrate cartilage

Lampreys possess unique types of cartilage in which elastin-like proteins are the dominant matrix component, whereas gnathostome cartilage is mainly composed of fibrillar collagen. Despite the differences in protein composition, the Sox-col2a1 genetic cascade was suggested to be conserved between lamprey pharyngeal cartilage and gnathostome cartilage. We examined whether the cascade is conserved in another type of lamprey cartilage, the trabecular cartilage. We found that SoxD and SoxE are expressed in both trabecular and pharyngeal cartilages. However, trabecular cartilage shows no clade A fibrillar collagen gene expression, including genes expressed in pharyngeal cartilage of this animal. On the basis of these observations, we propose that lampreys possess an ancestral type of cartilage that is similar to amphioxus gill cartilage, and in this respect, gnathostome cartilage can be regarded as derived for the loss of elastin-like protein as a cartilage component and recruitment of fibrillar collagen, which is included as a minor component in the ancestral cartilage, as the main component.

Microbial community associated with the colonial ascidian Cystodytes dellechiajei

The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.

Affinity purification of plasmid DNA by temperature‐triggered precipitation

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.

One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation.

The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordinated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of [(VPGVG)(2)(VPGKG)(VPGVG)(2)](21) were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP bio- polymers. Biopolymers charged with Ni(2+) were able to interact with a His tag on the target proteins based on metal coordination chemistry. Purifications of two His-tagged enzymes, beta-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the utility of this general method and over 85% recovery was observed in both cases. The bound enzymes were easily released by addition of either EDTA or imidazole. The recovered ELPs were reused four times with no observable decrease in the purification performance.

Design and Biosynthesis of Elastin-like Artificial Extracellular Matrix Proteins Containing Periodically Spaced Fibronectin CS5 Domains

Synthetic polymers have attained a dominant position in materials science and technology largely on the basis of their excellent physical and mechanical properties. The more subtle chemical and biological properties of natural polymers, especially of the proteins and nucleic acids, have been difficult to capture in synthetic macromolecular materials, in part because these properties arise from microstructural features that cannot be controlled in statistical polymerization processes. We describe herein the use of artificial genes to direct the synthesis of polymers of precisely controlled architecture, in which biological function specifically, the capacity to support attachment of vascular endothelial cellsis the primary object of the design. A long-term objective of this work is the development of improved materials for the regeneration, replacement or repair of vascular tissue.

A Family of Non–Collagen-Based Cartilages in the Skeleton of the Sea Lamprey, Petromyzon marinus

Cartilage structures of the sea lamprey, Petromyzon marinus, have previously been reported to stain by the Verhoeff method for elastin and to have a morphology similar to that of elastic cartilage of higher vertebrates. Earlier we showed that lamprin, the major matrix protein of lamprey annular cartilage, although not identical to elastin, cross-reacted with antibodies to elastin and shared significant sequence and structural similarities. Here we demonstrate that the majority of the skeletal cartilage of the lamprey is noncollagenous in character, remaining intact after treatment of the tissues with cyanogen bromide. However, unlike annular and neurocranial cartilages, which consist mainly of lamprin, evidence from amino acid compositions, immunohistochemistry, and western and northern blotting indicates that the major matrix protein of branchial and pericardial cartilage is not lamprin, but a second cyanogen bromide-insoluble protein or group of proteins with similarities to elastin. These results suggest that the skeletal cartilage of lower vertebrates consists of a family of noncollagenous, elastin-like proteins. The relationships among members of this family of cartilaginous proteins and between these proteins and vertebrate elastins, and the implications of these relationships for understanding the evolutionary lineage of elastin will await further characterization of these proteins.

Microscopic anatomy and histochemistry of the crayfish parasite Psorospermium haeckeli

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 21:79-90 (1995) - doi:10.3354/dao021079 Microscopic anatomy and histochemistry of the crayfish parasite Psorospermium haeckeli Vogt G, Rug M ABSTRACT: Psorospermium haeckeli, a common parasite of the European noble crayfish Astacus astacus, occurs as an egg-shaped spore of ca 100 x 60 x 60 um, particularly within the collagenous layer of the thoracic arteries and the surrounding subepidermal connective tissue. Smaller numbers are also found within the connective tissues of virtually all organs. The morphology and histology of these spores was investigated by microscopic techniques and the composition of their major structural components was identified by histochemistry. Each spore is enveloped by a layer of connective tissue which is assumed to derive from the host. It varies in thickness and composition among the infected organs. The spore proper consists of a 3-layered shell enclosing many globules. The outer shell layer is composed of solid, collagen- and elastin-negative, proteinaceous plates separated by sutures. The medial shell layer appears less compact and includes glycoproteins or neutral mucopolysaccharides. The inner layer is composed of 2 membranous structures including glycoproteins, glycolipids, and collagen- and elastin-like proteins. The interior of the spore contains lipid globules of variable size, intensely staining nonlipid globules with crystalline and vacuolar inclusions, and 2 closely associated nuclei. In most of the crayfish investigated some of the spores were encapsulated by haemocytes and melanized. KEY WORDS: Psorospermium haeckeli . Parasite . Astacus astacus . Crayfish . Microscopic anatomy . Histochemistry Full text in pdf format NextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 21, No. 2. Publication date: March 30, 1995 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1995 Inter-Research.

Composition of Normal and Pathological Cutaneous Elastin

ELASTIN, a fibrous protein invariably associated with collagen1,2, has been quantitatively measured in a variety of mammalian tissues3,4. The amino-acid composition of elastin prepared from various animal sources including aorta5, lung6 and ligamentum nuchae7 has been reported and the elastin content of human dermis has been determined8,9. The purpose of this work was to determine tho amino-acid composition of human dermal elastin and compare it with that of the elastin-like proteins found in increased amounts in chronically sun-damaged skin (actinic elastosis) and pseudoxanthoma elasticum8.