scholarly journals Lipid sponge droplets as programmable synthetic organelles

2020 ◽  
Vol 117 (31) ◽  
pp. 18206-18215
Author(s):  
Ahanjit Bhattacharya ◽  
Henrike Niederholtmeyer ◽  
Kira A. Podolsky ◽  
Rupak Bhattacharya ◽  
Jing-Jin Song ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Spatial Organization ◽  
Specific Binding ◽  
Reaction Rates ◽  
Nanoporous Structure ◽  
Single Chain ◽  
Reaction Systems ◽  
Biologically Relevant ◽  
Structure Lipid ◽  
Sponge Phase

Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here, we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and nonionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the colocalization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems.

scholarly journals Lipid Sponge Droplets as Programmable Synthetic Organelles

2020 ◽  
Author(s):  
Ahanjit Bhattacharya ◽  
Henrike Niederholtmeyer ◽  
Kira A. Podolsky ◽  
Rupak Bhattacharya ◽  
Jing-Jin Song ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Spatial Organization ◽  
Nanoporous Structure ◽  
Enzymatic Reactions ◽  
Single Chain ◽  
Reaction Systems ◽  
Dense Membrane ◽  
Synthetic Reaction ◽  
Sponge Phase ◽  
Membrane Networks

AbstractLiving cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and non-ionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems.Significance statementOrganelles spatially and temporally orchestrate biochemical reactions in a cell to a degree of precision that is still unattainable in synthetic reaction systems. Additionally, organelles such as the endoplasmic reticulum (ER) contain highly interconnected and dense membrane networks that provide large reaction spaces for both transmembrane and soluble enzymes. We present lipid sponge droplets to emulate the functions of organelles such as the ER. We demonstrate that lipid sponge droplets can be programmed to internally concentrate specific proteins, host and accelerate biochemical transformations, and to rapidly and reversibly sequester and release proteins to control enzymatic reactions. The self-assembled and programmable nature of lipid sponge droplets will facilitate the integration of complex functions for bottom up synthetic biology.

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

scholarly journals Polydopamine as an Efficient Polymer to Prepare Biologically Relevant Supported Lipid Bilayers

2015 ◽  
Vol 108 (2) ◽  
pp. 542a
Author(s):  
Souryvanh Nirasay ◽  
Antonella Badia ◽  
Jerome P. Claverie ◽  
Isabelle Marcotte
Keyword(s):  
Lipid Bilayers ◽  
Supported Lipid Bilayers ◽  
Biologically Relevant

Conductive AFM: Probing Nano-scale Electrical Properties of Model Cell Membranes

2012 ◽  
Vol 1465 ◽  
Author(s):  
Paul Farrar ◽  
Del Atkinson ◽  
Andrew J. Gallant
Keyword(s):  
Lipid Bilayers ◽  
Electrical Contact ◽  
Pyrolytic Graphite ◽  
Elastic Response ◽  
Electrical Measurements ◽  
Contact Mode ◽  
Biologically Relevant ◽  
Atomic Force ◽  
Current Voltage ◽  
Model Cell

ABSTRACTBiologically relevant lipid bilayers supported on highly ordered pyrolytic graphite (HOPG) were probed both mechanically and electrically with a Conductive Atomic Force Microscope (C-AFM) capable of measuring ultra-low currents. Results show that these membranes undergo an elastic response up to 26 nN on average when compressed with an AFM tip. Measuring the films with a low contact force demonstrates that contact mode AFM can be used repeatedly to image without damaging the film. Based on current-voltage measurements made with the C-AFM, it is shown that apparently high resistances seen for the films could be the result of variable electrical contact between the tip and surface. As a result, the paper proposes that the deflection of the cantilever should always be measured in order to ensure knowledge of the location of the tip during all electrical measurements.

Peptide discs as precursors of biologically relevant supported lipid bilayers

2021 ◽  
Vol 585 ◽  
pp. 376-385
Author(s):  
Alessandra Luchini ◽  
Federica Sebastiani ◽  
Frederik Grønbæk Tidemand ◽  
Krishna Chaithanya Batchu ◽  
Mario Campana ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Supported Lipid Bilayers ◽  
Biologically Relevant

Abstract 1033: Simultaneous Clot-targeted Factor Xa Inhibition And Selective Blockade Of Activated GPIIb/IIIa On Platelets Results In Delayed But Potent Antithrombotic Effects Without Bleeding Time Prolongation.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Christoph E Hagemeyer ◽  
Steffen U Eisenhardt ◽  
Nicole Bassler ◽  
Patrick Stoll ◽  
Meike Schwarz ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Specific Binding ◽  
Factor Xa ◽  
Single Chain ◽  
Occlusion Time ◽  
Selective Blockade ◽  
Thrombosis Model ◽  
Fibrinogen Binding ◽  
Antithrombotic Effects

Background: We generated phage-display-derived anti-GPIIb/IIIa single-chain antibodies (e.g. scFv SCE5) that specifically bind to the activated GPIIb/IIIa only and thus specifically block activated platelets only. ScFv SCE5 demonstrates strong antithrombotic potency, comparable to the conformation-unspecific blockers tirofiban and eptifibatide. In contrast bleeding times were not prolonged with scFv SCE5. Here we now use the possibility to add effector molecules using molecular biology methods. The highly potent anticoagulant TAP (tick anticoagulant peptide), which is a direct factor Xa (fXa) inhibitor, was used as an effector molecule. Methods and Results: We genetically fused the activation-specific scFv with TAP, expressed the constructs in E.coli and purified the 39 kDa protein via its Histag binding to Nickel beads. Specific binding of the fusion molecules MA2/SCE5-TAP and strong inhibition of fibrinogen binding was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. Prolongation in occlusion time with SCE5-TAP was significantly stronger compared to SCE5 alone, recombinant TAP, non-binding mut-scFv-TAP as well as the clinical used drugs enoxaparine and eptifibatide. In contrast to the other anticoagulants tested, bleeding time was not prolonged by SCE5-TAP. Flow experiments studying platelet adhesion on collagen revealed a possible mechanism for the unique finding of a fully normal bleeding time: LIBS exposure on adhering platelets and as such the anticoagulative targeting potency of SCE5-TAP was delayed until considerable layers of platelets were deposited. Conclusions: The combination of activation-specific GPIIb/IIIa blockade and fXa inhibition in one clot-targeted molecule further improves in-vivo antithrombotic efficiency without causing any bleeding time prolongation. The delay of the observed targeting effect may allow a sealing of injuries with platelet layers but may be in time for the prevention of occlusive platelet aggregates. The described blockers represent a new type of highly selective drugs that warrant further clinical development.

scholarly journals Coarse-graining via EDP-convergence for linear fast-slow reaction systems

2020 ◽  
Vol 30 (09) ◽  
pp. 1765-1807 ◽  
Author(s):  
Alexander Mielke ◽  
Artur Stephan
Keyword(s):  
Detailed Balance ◽  
Reaction System ◽  
Gradient Flow ◽  
Coarse Graining ◽  
Reaction Rates ◽  
Coarse Grained ◽  
Gradient Structure ◽  
Reaction Systems ◽  
Fast Reactions ◽  
Finite State

We consider linear reaction systems with slow and fast reactions, which can be interpreted as master equations or Kolmogorov forward equations for Markov processes on a finite state space. We investigate their limit behavior if the fast reaction rates tend to infinity, which leads to a coarse-grained model where the fast reactions create microscopically equilibrated clusters, while the exchange mass between the clusters occurs on the slow time scale. Assuming detailed balance the reaction system can be written as a gradient flow with respect to the relative entropy. Focusing on the physically relevant cosh-type gradient structure we show how an effective limit gradient structure can be rigorously derived and that the coarse-grained equation again has a cosh-type gradient structure. We obtain the strongest version of convergence in the sense of the Energy-Dissipation Principle (EDP), namely EDP-convergence with tilting.

scholarly journals Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wiggert J. Altenburg ◽  
N. Amy Yewdall ◽  
Daan F. M. Vervoort ◽  
Marleen H. M. E. van Stevendaal ◽  
Alexander F. Mason ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Nitrilotriacetic Acid ◽  
Binding Motif ◽  
Biologically Relevant ◽  
Enzyme Cascade ◽  
Synthetic Cell ◽  
Synthetic Cells ◽  
Membrane Bound ◽  
High Concentrations ◽  
High Level

AbstractThe cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

scholarly journals Generation of antibody-based therapeutics targeting the idiotype of B-cell malignancies

2018 ◽  
Vol 2 (1) ◽  
pp. 12-21
Author(s):  
Emily Weiss ◽  
Robert Sarnovsky ◽  
Mitchell Ho ◽  
Evgeny Arons ◽  
Robert Kreitman ◽  
...  
Keyword(s):  
B Cell ◽  
Specific Binding ◽  
Phage Library ◽  
Selection Strategy ◽  
Proof Of Concept ◽  
Single Chain ◽  
Human Igg1 ◽  
Specificity Of Binding ◽  
Complementarity Determining Regions ◽  
Phage Selection

ABSTRACT Background A feature of many B-cell tumors is a surface-expressed immunoglobulin (sIg). The complementarity-determining regions (CDRs) of the sIg, termed the ‘idiotype’, are unique to each tumor. We report on a phage selection strategy to generate anti-idiotype therapeutics that reacts with sIg CDR3 sequences; the MEC1 B-cell tumor line was used as proof of concept. Methods To create a mimetic of the MEC1 idiotype, CDR3 sequences from heavy and light chains of the sIg were grafted into a single chain variable fragment (scFv) framework scaffold. Using the Tomlinson I phage library of human scFvs, we enriched for binders to MEC1 CDR3 sequences over unrelated CDR3 sequences. Results By ELISA we identified 10 binder phages. Of these, five were sequenced, found to be unique and characterized further. By flow cytometry each of the five phages bound to MEC1 cells, albeit with different patterns of reactivity. To establish specificity of binding and utility, the scFv sequences from two of these binders (phages 1 and 7) were converted into antibody-toxin fusion proteins (immunotoxins) and also cloned into a human IgG1 expression vector. Binders 1 and 7 immunotoxins exhibited specific killing of MEC1 cells with little toxicity for non-target B-cell lines. The full-length antibody recreated from the binder-1 scFv also exhibited specific binding. Conclusion Our results establish the utility of using engrafted CDR3 sequences for selecting phage that recognize the idiotype of B-cell tumors.

scholarly journals Expression, Purification, and Characterization of Anti-Zika virus Envelope Protein: Polyclonal and Chicken-Derived Single Chain Variable Fragment Antibodies

2020 ◽  
Vol 21 (2) ◽  
pp. 492 ◽  
Author(s):  
Pharaoh Fellow Mwale ◽  
Chi-Hsin Lee ◽  
Liang-Tzung Lin ◽  
Sy-Jye Leu ◽  
Yun-Ju Huang ◽  
...  
Keyword(s):  
Zika Virus ◽  
Envelope Protein ◽  
Therapeutic Potential ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Virus Envelope ◽  
Phage Display Technology ◽  
High Level

Zika virus (ZIKV) is a new and emerging virus that has caused outbreaks worldwide. The virus has been linked to congenital neurological malformations in neonates and Guillain–Barré syndrome in adults. Currently there are no effective vaccines available. As a result, there is a great need for ZIKV treatment. In this study, we developed single chain variable fragment (scFv) antibodies that target the ZIKV envelope protein using phage display technology. We first induced an immune response in white leghorn laying hens against the ZIKV envelope (E) protein. Chickens were immunized and polyclonal immunoglobulin yolk (IgY) antibodies were extracted from egg yolks. A high-level titer of anti-ZIKV_E IgY antibodies was detected using enzyme-linked immunosorbent assay (ELISA) after the third immunization. The titer persisted for at least 9 weeks. We constructed two antibody libraries that contained 5.3 × 106 and 4.5 × 106 transformants. After biopanning, an ELISA phage assay confirmed the enrichment of specific clones. We randomly selected 26 clones that expressed ZIKV scFv antibodies and classified them into two groups, short-linker and long-linker. Of these, four showed specific binding activities toward ZIKV_E proteins. These data suggest that the polyclonal and monoclonal scFv antibodies have the diagnostic or therapeutic potential for ZIKV.

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