F. Javier Arias

Immunomodulatory Nanoparticles from Elastin-Like Recombinamers: Single-Molecules for Tuberculosis Vaccine Development

This study investigates both the physicochemical properties and immunogenicity of a genetically engineered elastin-like block corecombinamer (ELbcR) containing a major membrane protein sequence from Mycobacterium tuberculosis. The recombinant production of this ELbcR allows the production of large quantities of safe, antigenic particle-based constructs that directly and reversibly self-assemble into highly biocompatible, multivalent, monodisperse, and stable nanovesicles with a diameter of 55 nm from the same gene product using a highly efficient and cost-effective inverse transition cycling (ITC) procedure. The compositional complexity of these vesicles is retained after secondary processes such as endotoxin removal, sterilization, and lyophilization. An initial pro-chemotactic cytokine response (IL-1β) followed by a pro-Th2/IL-5 response was observed in mice plasma following subcutaneous administration of the antigen-loaded nanovesicles in mice. This biphasic model of cytokine production was coupled with humoral isotype switching from IgM- to IgG-specific antibodies against the antigen, which was only observed in the presence of both the antigen and the polymer in the same construct and in the absence of additional adjuvants.

Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae).

We have studied the distribution of the protein synthesis inhibitory activity in the tissues of Saponaria officinalis L. (Caryophyllaceae). Seven major saporins, ribosome-inactivating proteins, were purified to apparent homogeneity from leaves, roots and seeds using a new procedure of RIPs isolation including ion-exchange and hydrophobic chromatography. They all catalysed the depurination of rat liver ribosomes, which generate the Endos diagnostic rRNA fragment upon treatment with acid aniline, thus indicating that A4324 from the 28S rRNA has been released (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). The molecular mass of saporins by SDS-PAGE ranged between 30.2 and 31.6 kDa and by gel-filtration between 27.5 and 30.1 kDa. Amino acid composition and amino-terminal amino acid sequence indicate that all saporins may be considered isoforms. Only two saporins present in roots were glycosylated (SO-R1 and SO-R3). All saporins are very active on cell-free translation systems derived from rabbit reticulocyte lysates, rat liver, Triticum aestivum L., Cucumis sativus L. and Vicia sativa L. However, they are poor inhibitors of an Escherichia coli translation system. They inhibit protein synthesis in HeLa, BeWo and NB 100 cells, HeLa cells being the most resistant. The enzymatic activity of at least one saporin isoform was dependent on magnesium concentration in the standard rat liver cell-free system.

Isolation and partial characterization of nigrin b, a non-toxic novel type 2 ribosome-inactivating protein from the bark ofSambucus nigra L.

The bark ofSambucus nigra L. contains a non-toxic novel type 2 ribosome-inactivating protein that we named nigrin b.In vitro, nigrin b strongly inhibited mammalian protein synthesis but did not affect plant nor bacterial protein synthesis. The protein (Mr 58 000) contains two subunits, A (Mr 26 000) and B (Mr 32 000); linked by disulphide bridge(s). Nigrin b was found to be an rRNA N-glycosidase of the rRNA of intact mammalian ribosomes and shares a very good N-terminal amino-acid sequence homology with the anti-HIV-1 proteins TAP 29 and trichosanthin.

Biofunctional design of elastin-like polymers for advanced applications in nanobiotechnology

Elastin-like recombinant protein polymers are a new family of polymers which are captivating the attention of a broad audience ranging from nanotechnologists to biomaterials and more basic scientists. This is due to the extraordinary confluence of different properties shown by this kind of material that are not found together in other polymer systems. Elastin-like polymers are extraordinarily biocompatible, acutely smart and show uncommon self-assembling capabilities. Additionally, they are highly versatile, since these properties can be tuned and expanded in many different ways by substituting the amino acids of the dominating repeating peptide or by inserting, in the polymer architecture, (bio)functional domains extracted from other natural proteins or de novo designs. Recently, the potential shown by elastin-like polymers has, in addition, been boosted and amplified by the use of recombinant DNA technologies. By this means, complex molecular designs and extreme control over the amino-acid sequence can be attained. Nowadays, the degree of complexity and control shown by the elastin-like protein polymers is well beyond the reach of even the most advanced polymer chemistry technologies. This will open new possibilities in obtaining synthetic advanced bio- and nanomaterials. This review explores the present development of elastin-like protein polymers, with a particular emphasis for biomedical uses, along with some future directions that this field will likely explore in the near future.

Synthesis and Characterization of Macroporous Thermosensitive Hydrogels from Recombinant Elastin-Like Polymers

Multifunctional bioactive chemically cross-linked elastin-like polymers (ELPs) have been prepared as three-dimensional scaffolds for tissue engineering. The salt-leaching/gas-foaming technique was found suitable to prepare highly porous biodegradable hydrogels based on this novel material type. The porosity can be controlled by the amount of sodium hydrogen carbonate incorporated during the cross-linking reaction, whereas the mean pore size is determined by the salt particle size. The gas-foaming process, which involves immersion in a citric acid solution after the cross-linking, facilitates pore interconnectivity and allows a grooved surface essential for cell colonization. Due to the thermoresponsive nature of the ELPs, their physical properties are strongly influenced by the temperature of the aqueous medium. The feasibility to obtain tridimensional scaffolds for tissue engineering has been studied by testing the adhesion and spreading of endothelial cells into the porous ELP hydrogels. The methods and structures described herein provide a starting point for the design and synthesis of macroporous multifunctional elastin-like hydrogels with potential broad applicability.

Influence of the Amino-Acid Sequence on the Inverse Temperature Transition of Elastin-Like Polymers

This work explores the dependence of the inverse temperature transition of elastin-like polymers (ELPs) on the amino-acid sequence, i.e., the amino-acid arrangement along the macromolecule and the resulting linear distribution of the physical properties (mainly polarity) derived from it. The hypothesis of this work is that, in addition to mean polarity and molecular mass, the given amino-acid sequence, or its equivalent--the way in which polarity is arranged along the molecule--is also relevant for determining the transition temperature and the latent heat of that transition. To test this hypothesis, a set of linear and di- and triblock ELP copolymers were designed and produced as recombinant proteins. The absolute sequence control provided by recombinant technologies allows the effect of the amino-acid arrangement to be isolated while keeping the molecular mass or mean polarity under strict control. The selected block copolymers were made of two different ELPs: one exhibiting temperature and pH responsiveness, and one exhibiting temperature responsiveness only. By changing the arrangement and length of the blocks while keeping other parameters, such as the molecular mass or mean polarity, constant, we were able to show that the sequence plays a key role in the smart behavior of ELPs.

Tailored recombinant elastin-like polymers for advanced biomedical and nano(bio)technological applications

The genetic engineering of protein-based polymers is a method that enables, in an easy way, the design of complex and highly functional macromolecules. As examples of this approach, different molecular designs are presented, with increasing degree of complexity, showing how the controlled increase in their complexity yields (multi)functional materials with more selected and sophisticated properties. The simplest designs show interesting properties already, but the adequate introduction of given chemical functions along the polymer chain provides an opportunity to expand the range of properties to enhanced smart behavior and self-assembly. Finally, examples are given where those molecular designs further incorporate selected bioactivities in order to develop materials for the most cutting edge applications in biomedicine and nano(bio)technology.

Genetic Engineering of Protein-Based Polymers: The Example of Elastinlike Polymers

In spite of the enormous possibilities of macromolecules as key elements in developing advancedmaterials with increased functionality and complexity, the success in this development is often limitedby the randomness associated with polymer synthesis and the exponential increase in technical difficultiescaused by the attempt to reach a sufficiently high degree of complexity in the molecular design.This paper describes a new approach in the design of complex and highly functional macromolecules,the genetic engineering of protein-based macromolecules. The exploitation of the efficient machineryof protein synthesis in living cells opens a path to obtain extremely well-defined and complexmacromolecules. Different molecular designs are presented, with increasing degree of complexity,showing how the controlled increase in their complexity yields (multi)functional materials with moreselect and sophisticated properties. The simplest designs show interesting properties already, butthe adequate introduction of given chemical functions along the polymer chain presents an opportunityto expand the range of properties to enhanced smart behavior and self-assembly. Finally, examplesare given where those molecular designs further incorporate selected bioactivities in order to developmaterials for the most cutting-edge applications in the field of biomedicine and nano(bio)technology.

Molecular mechanism of inhibition of mammalian protein synthesis by some four-chain agglutinins. Proposal of an extended classification of plant ribosome-inactivating proteins (rRNA N-glycosidases).

The four chain agglutinins from Abrus precatorius, Viscum album and Ricinus communis promote depurination of the 28 S rRNA from rabbit reticulocyte ribosomes characteristic of the common ribosome‐inactivating proteins (RIPs). These agglutinins inhibited mammalian protein synthesis at nanomolar concentrations but they do not affect plant protein synthesis under the same conditions. Therefore, they should also be considered as true RIPs but of a new class, the four‐chain RIPs. An extended classification of RIPs is presented based on the former one from Stirpe et al. [Bio/technology 10 (1992) 405‐412].

Cusativin, a new cytidine-specific ribonuclease accumulated in seeds of Cucumis sativus L.

Dry seeds of Cucumis sativus L. were found to contain a heat-sensitive endoribonuclease of a novel type which we have named cusativin. It was purified to apparent electrophoretic homogeneity by chromatography through S-Sepharose Fast Flow, Sephadex G-75, CM-Sepharose, Superdex 75-FPLC (fast protein liquid chromatography) and Mono S-FPLC. It is a single unglycosylated polypeptide chain with an apparent molecular mass (Mr) of 22900. Polyclonal anti-cusativin antibodies raised in rabbits only reacted with melonin, the translation inhibitor from Cucumis melo L. Functional, Western blot and enzyme-linked immunosorbent assay (ELISA) analyses indicated that cusativin is present in the coat and cotyledons of dry seeds, but not in embryonic axes. Cusativin is accumulated in maturing seeds. By contrast, after seed germination there is degradation of the cusativin present in cotyledons but not that present in the seed coat. The preference of cusativin for polynucleotide cleavage was poly(C)≫poly(A) acids, poly(U) and poly(G) being unaffected by cusativin. Under the denaturing conditions used for RNA sequencing, cusativin acted only on poly(C). Cusativin proved to be useful for RNA sequencing, in particular, complementing the data obtained with RNase CL3. Cusativin represents a new class of plant RNase and, as far as we are aware, is the first plant enzyme that shows cleavage specificity for cytidine under the denaturing conditions of RNA sequencing.