The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Andreas Pluckthun - One of the best experts on this subject based on the ideXlab platform.

  • Engineered Proteins with desired specificity darpins other alternative scaffolds and bispecific iggs
    Current Opinion in Structural Biology, 2014
    Co-Authors: Christian Jost, Andreas Pluckthun
    Abstract:

    Specific binding Proteins have become essential for diagnostic and therapeutic applications, and traditionally these have been antibodies. Nowadays an increasing number of alternative scaffolds have joined these ranks. These additional folds have raised a lot of interest and expectations within the last decade. It appears that they have come of age and caught up with antibodies in many fields of applications. The last years have seen an exploration of possibilities in research, diagnostics and therapy. Some scaffolds have received further improvements broadening their fields of application, while others have started to occupy their respective niche. Protein engineering, the prerequisite for the advent of all alternative scaffolds, remains the driving force in this process, for both non-immunoglobulins and immunoglobulins alike.

  • Engineered Proteins as specific binding reagents
    Current Opinion in Biotechnology, 2005
    Co-Authors: Kaspar H Binz, Andreas Pluckthun
    Abstract:

    Over the past 30 years, monoclonal antibodies have become the standard binding Proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding Proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

Sarah C Heilshorn - One of the best experts on this subject based on the ideXlab platform.

  • covalently adaptable elastin like protein hyaluronic acid elp ha hybrid hydrogels with secondary thermoresponsive crosslinking for injectable stem cell delivery
    Advanced Functional Materials, 2017
    Co-Authors: Huiyuan Wang, Danqing Zhu, Alexandra Paul, Lei Cai, Annika Enejder, Fan Yang, Sarah C Heilshorn
    Abstract:

    Shear-thinning, self-healing hydrogels are promising vehicles for therapeutic cargo delivery due to their ability to be injected using minimally invasive surgical procedures. An injectable hydrogel using a novel combination of dynamic covalent crosslinking with thermoresponsive Engineered Proteins is presented. Ex situ at room temperature, rapid gelation occurs through dynamic covalent hydrazone bonds by simply mixing two components: hydrazine-modified elastin-like protein (ELP) and aldehyde-modified hyaluronic acid. This hydrogel provides significant mechanical protection to encapsulated human mesenchymal stem cells during syringe needle injection and rapidly recovers after injection to retain the cells homogeneously within a 3D environment. In situ, the ELP undergoes a thermal phase transition, as confirmed by coherent anti-Stokes Raman scattering microscopy observation of dense ELP thermal aggregates. The formation of the secondary network reinforces the hydrogel and results in a tenfold slower erosion rate compared to a control hydrogel without secondary thermal crosslinking. This improved structural integrity enables cell culture for three weeks postinjection, and encapsulated cells maintain their ability to differentiate into multiple lineages, including chondrogenic, adipogenic, and osteogenic cell types. Together, these data demonstrate the promising potential of ELP-HA hydrogels for injectable stem cell transplantation and tissue regeneration.

  • design of three dimensional Engineered protein hydrogels for tailored control of neurite growth
    Acta Biomaterialia, 2013
    Co-Authors: Kyle J Lampe, Alexander L Antaris, Sarah C Heilshorn
    Abstract:

    The design of bioactive materials allows tailored studies probing cell–biomaterial interactions, however, relatively few studies have examined the effects of ligand density and material stiffness on neurite growth in three-dimensions. Elastin-like Proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within threedimensional (3-D) materials. To promote neurite out-growth and better understand the effects of common biomaterial design parameters on neuronal cultures we here focused on the cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of Engineered Proteins these 3-D ELP hydrogels enabled decoupled investigations into the effects of biomechanics and biochemistry on neurite out-growth from dorsal root ganglia. Increasing the cell-adhesive RGD ligand density from 0 to 1.9 � 10 7 ligands lm

  • designer protein based scaffolds for neural tissue engineering
    International Conference of the IEEE Engineering in Medicine and Biology Society, 2009
    Co-Authors: Karin S Straley, Sarah C Heilshorn
    Abstract:

    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 family of biomimetic scaffolds composed of chemically crosslinked, elastin-like Proteins designed to support neural regeneration through a combination of cell adhesion and cell-induced degradation and remodeling. Through use of a modular protein-design strategy, a range of biomaterials is created that allows independent tuning over the initial elastic modulus, degradation rate, cell adhesivity, and neurite outgrowth. By combining these Engineered Proteins into composite structures, biomaterials are created with 3D patterns that emerge over time in response to cell-secreted enzymes. These dynamic 3D structures enable the delivery of multiple drugs with precise spatial and temporal resolution and also enable the design of biomaterials that adapt to changing scaffold needs.

  • design and adsorption of modular Engineered Proteins to prepare customized neuron compatible coatings
    Frontiers in Neuroengineering, 2009
    Co-Authors: Karin S Straley, Sarah C Heilshorn
    Abstract:

    Neural prosthetic implants are currently being developed for the treatment and study of both peripheral and central nervous system disorders. Effective integration of these devices upon implantation is a critical hurdle to achieving function. As a result, much attention has been directed towards the development of biocompatible coatings that prolong their in vivo lifespan. In this work, we present a novel approach to fabricate such coatings, which specifically involves the use of surface-adsorbed, nanoscale-designed protein polymers to prepare reproducible, customized surfaces. A nanoscale modular design strategy was employed to synthesize six Engineered, recombinant Proteins intended to mimic aspects of the extracellular matrix Proteins fibronectin, laminin, and elastin as well as the cell-cell adhesive protein NCAM (neural cell adhesion molecule). Physical adsorption isotherms were experimentally determined for these Engineered Proteins, allowing for direct calculation of the available ligand density present on coated surfaces. As confirmation that ligand density in these Engineered systems impacts neuronal cell behavior, we demonstrate that increasing the density of fibronectin-derived RGD ligands on coated surfaces while maintaining uniform protein surface coverage results in enhanced neurite extension of PC-12 cells. Therefore, this Engineered protein adsorption approach allows for the facile preparation of tunable, quantifiable, and reproducible surfaces for in vitro studies of cell-ligand interactions and for potential application as coatings on neural implants.

  • Gradient lithography of Engineered Proteins to fabricate 2D and 3D cell culture microenvironments
    Biomedical Microdevices, 2009
    Co-Authors: Sheng Wang, Sarah C Heilshorn, Cheryl Wong Po Foo, Ajithkumar Warrier, Xiang Zhang
    Abstract:

    Spatial patterning of Proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research.

Oliver Griesbeck - One of the best experts on this subject based on the ideXlab platform.

  • targeted in situ protein diversification and intra organelle validation in mammalian cells
    Chemistry & Biology, 2020
    Co-Authors: Mutlu Erdogan, Arne Fabritius, Jerome Basquin, Oliver Griesbeck
    Abstract:

    Summary Engineered Proteins must be phenotypically selected for function in the appropriate physiological context. Here, we present a versatile approach that allows generating panels of mammalian cells that express diversified heterologous protein libraries in the cytosol or subcellular compartments under stable conditions and in a single-variant-per-cell manner. To this end we adapt CRISPR/Cas9 editing technology to diversify targeted stretches of a protein of interest in situ. We demonstrate the utility of the approach by in situ engineering and intra-lysosome specific selection of an extremely pH-resistant long Stokes shift red fluorescent protein variant. Tailoring properties to specific conditions of cellular sub-compartments or organelles of mammalian cells can be an important asset to optimize various Proteins, protein-based tools, and biosensors for distinct functions.

Jim Warwicker - One of the best experts on this subject based on the ideXlab platform.

  • modelling of ph dependence to develop a strategy for stabilising mabs at acidic steps in production
    Computational and structural biotechnology journal, 2020
    Co-Authors: Max Hebditch, Ryan Kean, Jim Warwicker
    Abstract:

    Engineered Proteins are increasingly being required to function or pass through environmental stresses for which the underlying protein has not evolved. A major example in health are antibody therapeutics, where a low pH step is used for purification and viral inactivation. In order to develop a computational model for analysis of pH-stability, predictions are compared with experimental data for the relative pH-sensitivities of antibody domains. The model is then applied to proteases that have evolved to be functional in an acid environment, showing a clear signature for low pH-dependence of stability in the neutral to acidic pH region, largely through reduction of salt-bridges. Interestingly, an extensively acidic protein surface can maintain contribution to structural stabilisation at acidic pH through replacement of basic sidechains with polar, hydrogen-bonding groups. These observations form a design principle for engineering acid-stable Proteins.

  • modelling of ph dependence to develop a strategy for stabilising mabs at acidic steps in production
    bioRxiv, 2019
    Co-Authors: Max Hebditch, Ryan Kean, Jim Warwicker
    Abstract:

    Engineered Proteins are increasingly being required to function or pass through environmental stresses for which the underlying protein has not evolved. A major example in health are antibody therapeutics, where a low pH step is used for purification and viral clearance. In order to develop a computational model for analysis of pH-stability, predictions are compared with experimental data for the relative pH-sensitivities of antibody domains. The model is then applied to proteases that have evolved to be functional in an acid environment, showing a clear signature for low pH-dependence of stability in the neutral to acidic pH region, largely through reduction of salt-bridges. Interestingly, an extensively acidic protein surface can maintain contribution to structural stabilisation at acidic pH through replacement of basic sidechains with polar, hydrogen-bonding groups. These observations form a design principle for engineering acid-stable Proteins.

Valder R Arruda - One of the best experts on this subject based on the ideXlab platform.

  • protein Engineered coagulation factors for hemophilia gene therapy
    Molecular therapy. Methods & clinical development, 2019
    Co-Authors: Benjamin J Samelsonjones, Valder R Arruda
    Abstract:

    Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies in either coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Recently, gene therapy clinical trials with adeno-associated virus (AAV) vectors and protein-Engineered transgenes, B-domain deleted (BDD) FVIII and FIX-Padua, have reported near-phenotypic cures in subjects with HA and HB, respectively. Here, we review the biology and the clinical development of FVIII-BDD and FIX-Padua as transgenes. We also examine alternative bioengineering strategies for FVIII and FIX, as well as the immunological challenges of these approaches. Other Engineered Proteins and their potential use in gene therapy for hemophilia with inhibitors are also discussed. Continued advancement of gene therapy for HA and HB using protein-Engineered transgenes has the potential to alleviate the substantial medical and psychosocial burdens of the disease.

  • Protein-Engineered Coagulation Factors for Hemophilia Gene Therapy
    Elsevier, 2019
    Co-Authors: Benjamin J. Samelson-jones, Valder R Arruda
    Abstract:

    Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies in either coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Recently, gene therapy clinical trials with adeno-associated virus (AAV) vectors and protein-Engineered transgenes, B-domain deleted (BDD) FVIII and FIX-Padua, have reported near-phenotypic cures in subjects with HA and HB, respectively. Here, we review the biology and the clinical development of FVIII-BDD and FIX-Padua as transgenes. We also examine alternative bioengineering strategies for FVIII and FIX, as well as the immunological challenges of these approaches. Other Engineered Proteins and their potential use in gene therapy for hemophilia with inhibitors are also discussed. Continued advancement of gene therapy for HA and HB using protein-Engineered transgenes has the potential to alleviate the substantial medical and psychosocial burdens of the disease. Keywords: Hemophilia A, hemophilia B, gene therapy, bioengineering, factor VIII, factor IX, factor IX Padua, B-domain delete factor VIII, immunogenicity, protein engineerin