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1.
Eur Phys J E Soft Matter ; 47(6): 37, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38829453

RESUMEN

In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.


Asunto(s)
Células Artificiales , Polímeros , Células Artificiales/química , Polímeros/química , Polímeros/síntesis química , Emulsiones/química , Cápsulas/química , Microfluídica/métodos , Agua/química , Técnicas Analíticas Microfluídicas , Proteínas/química
2.
Nat Commun ; 15(1): 5425, 2024 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-38926339

RESUMEN

Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society's most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales-molecular, circuit/network, cell/cell-free systems, biological communities, and societal-giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies. We present this framework, lessons learned, and inclusive teaching materials to allow its adaption to train the next generation of synthetic biologists.


Asunto(s)
Biología Sintética , Biología Sintética/educación , Biología Sintética/métodos , Humanos , Biotecnología/educación , Estudiantes/psicología
3.
bioRxiv ; 2024 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-38766162

RESUMEN

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive states are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here we describe a fully synthetic method to assay the functional properties of the polycystin subfamily of transient receptor potential (TRP) channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their membrane integration, orientation and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate other channels resistant to biophysical analysis and pharmacological characterization.

4.
Nat Commun ; 15(1): 3162, 2024 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-38605024

RESUMEN

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.


Asunto(s)
Células Artificiales , Proteínas de la Membrana , Membrana Celular/metabolismo , Membranas/metabolismo , Proteínas de la Membrana/metabolismo , Liposomas , Membrana Dobles de Lípidos/química
6.
ACS Synth Biol ; 13(1): 129-140, 2024 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-38150067

RESUMEN

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE.


Asunto(s)
Biosíntesis de Proteínas , Pliegue de Proteína , Biosíntesis de Proteínas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ribosomas/metabolismo , Péptidos/metabolismo , Lípidos
7.
Proc Natl Acad Sci U S A ; 120(19): e2218610120, 2023 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-37126679

RESUMEN

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step toward this goal, we investigated how bacterial two-component systems (TCSs) can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the TCS can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing.


Asunto(s)
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Regulación Bacteriana de la Expresión Génica , Proteínas de Unión al ADN/metabolismo
8.
Small Methods ; 7(12): e2201718, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37116099

RESUMEN

The surface modification of membrane-based nanoparticles, such as liposomes, polymersomes, and lipid nanoparticles, with targeting molecules, such as binding proteins, is an important step in the design of therapeutic materials. However, this modification can be costly and time-consuming, requiring cellular hosts for protein expression and lengthy purification and conjugation steps to attach proteins to the surface of nanocarriers, which ultimately limits the development of effective protein-conjugated nanocarriers. Here, the use of cell-free protein synthesis systems to rapidly create protein-conjugated membrane-based nanocarriers is demonstrated. Using this approach, multiple types of functional binding proteins, including affibodies, computationally designed proteins, and scFvs, can be cell-free expressed and conjugated to liposomes in one-pot. The technique can be expanded further to other nanoparticles, including polymersomes and lipid nanoparticles, and is amenable to multiple conjugation strategies, including surface attachment to and integration into nanoparticle membranes. Leveraging these methods, rapid design of bispecific artificial antigen presenting cells and enhanced delivery of lipid nanoparticle cargo in vitro is demonstrated. It is envisioned that this workflow will enable the rapid generation of membrane-based delivery systems and bolster our ability to create cell-mimetic therapeutics.


Asunto(s)
Liposomas , Nanopartículas , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química
9.
Biomacromolecules ; 24(4): 1574-1584, 2023 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-36943688

RESUMEN

The addition of both cell-targeting moieties and polyethylene glycol (PEG) to nanoparticle (NP) drug delivery systems is a standard approach to improve the biodistribution, specificity, and uptake of therapeutic cargo. The spatial presentation of these molecules affects avidity of the NP to target cells in part through an interplay between the local ligand concentration and the steric hindrance imposed by PEG molecules. Here, we show that lipid phase separation in nanoparticles can modulate liposome avidity by changing the proximity of PEG and targeting protein molecules on a nanoparticle surface. Using lipid-anchored nickel-nitrilotriacetic acid (Ni-NTA) as a model ligand, we demonstrate that the attachment of lipid anchored Ni-NTA and PEG molecules to distinct lipid domains in nanoparticles can enhance liposome binding to cancer cells by increasing ligand clustering and reducing steric hindrance. We then use this technique to enhance the binding of RGD-modified liposomes, which can bind to integrins overexpressed on many cancer cells. These results demonstrate the potential of lipid phase separation to modulate the spatial presentation of targeting and shielding molecules on lipid nanocarriers, offering a powerful tool to enhance the efficacy of NP drug delivery systems.


Asunto(s)
Liposomas , Polietilenglicoles , Liposomas/química , Ligandos , Distribución Tisular , Polietilenglicoles/química , Lípidos/química , Sistemas de Liberación de Medicamentos/métodos
10.
Trends Biotechnol ; 41(3): 276-277, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36646525

RESUMEN

Morsut et al. reported a synthetic receptor system, based on the natural Notch receptor, with customizable input and output functions. Their work on advanced receptor design expands the reach of synthetic receptor systems. Incorporating new protein design tools with better-understood membrane biophysics will create the next generation of engineered receptors.


Asunto(s)
Ingeniería de Proteínas , Receptores Artificiales
11.
Curr Opin Biotechnol ; 79: 102888, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36641905

RESUMEN

Vaccines are vital for protection against existing and emergent diseases. Current vaccine production strategies are limited by long production times, risky viral material, weak immunogenicity, and poor stability, ultimately restricting the safe or rapid production of vaccines for widespread utilization. Cell-free protein synthesis (CFPS) systems, which use extracted transcriptional and translational machinery from cells, are promising tools for vaccine production because they can rapidly produce proteins without the constraints of living cells, have a highly optimizable open system, and can be used for on-demand biomanufacturing. Here, we review how CFPS systems have been explored for the production of subunit, conjugate, virus-like particle (VLP), and membrane-augmented vaccines and as a tool in vaccine design. We also discuss efforts to address potential limitations with CFPS such as the presence of endotoxins, poor protein folding, reaction stability, and glycosylation to enable promising future vaccine design and production.


Asunto(s)
Vacunas de Partículas Similares a Virus , Desarrollo de Vacunas
12.
Sci Adv ; 9(1): eadd6605, 2023 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-36598992

RESUMEN

Cell-free systems have enabled the development of genetically encoded biosensors to detect a range of environmental and biological targets. Encapsulation of these systems in synthetic membranes to form artificial cells can reintroduce features of the cellular membrane, including molecular containment and selective permeability, to modulate cell-free sensing capabilities. Here, we demonstrate robust and tunable performance of a transcriptionally regulated, cell-free riboswitch encapsulated in lipid membranes, allowing the detection of fluoride, an environmentally important molecule. Sensor response can be tuned by varying membrane composition, and encapsulation protects from sensor degradation, facilitating detection in real-world samples. These sensors can detect fluoride using two types of genetically encoded outputs, enabling detection of fluoride at the Environmental Protection Agency maximum contaminant level of 0.2 millimolars. This work demonstrates the capacity of bilayer membranes to confer tunable permeability to encapsulated, genetically encoded sensors and establishes the feasibility of artificial cell platforms to detect environmentally relevant small molecules.

13.
ACS Synth Biol ; 12(2): 502-510, 2023 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-36651574

RESUMEN

Assembling transmembrane proteins on organic electronic materials is one promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. While transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs), incorporating functional and oriented transmembrane proteins into these structures remains challenging. Here, we demonstrate that cell-free expression systems allow for the one-step integration of an ion channel into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). Using the large conductance mechanosensitive channel (MscL) as a model ion channel, we demonstrate that MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts. This work serves as an important illustration of a rapidly assembled bioelectronic platform with a diverse array of downstream applications, including electrochemical sensing, physiological regulation, and screening of transmembrane protein modulators.


Asunto(s)
Técnicas Biosensibles , Membrana Dobles de Lípidos , Membrana Dobles de Lípidos/metabolismo , Canales Iónicos , Proteínas de la Membrana/metabolismo , Electrónica , Electrodos
15.
Biomacromolecules ; 23(11): 4756-4765, 2022 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-36318160

RESUMEN

Hybrid membranes assembled from biological lipids and synthetic polymers are a promising scaffold for the reconstitution and utilization of membrane proteins. Recent observations indicate that inclusion of small fractions of polymer in lipid membranes can improve protein folding and function, but the exact structural and physical changes a given polymer sequence imparts on a membrane often remain unclear. Here, we use all-atom molecular dynamics simulations to study the structure of hybrid membranes assembled from DOPC phospholipids and PEO-b-PBD diblock copolymers. We verified our computational model using new and existing experimental data and obtained a detailed picture of the polymer conformations in the lipid membrane that we can relate to changes in membrane elastic properties. We find that inclusion of low polymer fractions induces transient packing defects into the membrane. These packing defects act as insertion sites for two model peptides, and in this way, small amounts of polymer content in lipid membranes can lead to large increases in peptide insertion rates. Additionally, we report the peptide conformational space in both pure lipid and hybrid membranes. Both membranes support similar alpha helical peptide structures, exemplifying the biocompatibility of hybrid membranes.


Asunto(s)
Fosfolípidos , Polímeros , Polímeros/química , Membranas/metabolismo , Fosfolípidos/química , Membranas Artificiales , Péptidos , Membrana Dobles de Lípidos/química
16.
Small ; 18(19): e2200125, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35388947

RESUMEN

The ability of pathogens to develop drug resistance is a global health challenge. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody (mAb) therapies and vaccine-induced sera. Decoy nanoparticles-cell-mimicking particles that bind and inhibit virions-are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, quantitative understanding as to how design features impact performance of these therapeutics is lacking. To address this gap, this study presents a systematic, comparative evaluation of various biologically derived nanoscale vesicles, which may be particularly well suited to sustained or repeated administration in the clinic due to low toxicity, and investigates their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade mAbs and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.


Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Monoclonales/uso terapéutico , Antivirales , Humanos , Glicoproteína de la Espiga del Coronavirus/metabolismo
17.
Nano Lett ; 22(7): 2627-2634, 2022 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-35298184

RESUMEN

Ligand spatial presentation and density play important roles in signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Here, we harness lipid phase separation to spatially control the protein presentation on lipid vesicles. We use this system to improve the cytotoxicity of TNF-related apoptosis inducing ligand (TRAIL), a therapeutic anticancer protein. Vesicles with phase-separated TRAIL presentation induce more cell death in Jurkat cancer cells than vesicles with uniformly presented TRAIL, and cytotoxicity is dependent on TRAIL density. We assess this relationship in other cancer cell lines and demonstrate that phase-separated vesicles with TRAIL only enhance cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, is less sensitive to TRAIL density. This work demonstrates a rapid and accessible method to control protein conjugation and density on vesicles that can be adopted to other nanoparticle systems to improve receptor signaling by nanoparticles.


Asunto(s)
Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Ligando Inductor de Apoptosis Relacionado con TNF , Apoptosis , Línea Celular Tumoral , Humanos , Ligandos , Lípidos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología
18.
Methods Mol Biol ; 2433: 257-271, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34985750

RESUMEN

Hybrid membranes comprised of diblock copolymers, and phospholipids have gained interest due to their unique properties that result from blending natural and synthetic components. The integration of membrane proteins into these synthetic membranes is an important step towards creating biomembrane systems for uses such as artificial cellular systems, biosensors, and drug delivery vehicles. Here, we outline a technique to create hybrid membranes composed of phospholipids and diblock copolymers. Next, we describe how membrane proteins can be co-translationally integrated into hybrid lipid/polymer membranes using a cell-free reaction. We then outline a method to monitor insertion and folding of a membrane-embedded channel protein into the hybrid membrane using a fluorescent-protein reporter and dye release assay, respectively. This method is expected to be applicable for a wide range of membrane proteins that do not require chaperones for co-translational integration into vesicles and provides a generalized protocol for expressing a membrane protein into a membrane mimetic.


Asunto(s)
Células Artificiales , Polímeros , Proteínas de la Membrana , Fosfolípidos
19.
bioRxiv ; 2021 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-34909773

RESUMEN

The ability of pathogens to develop drug resistance is a global health challenge. The SARS-CoV-2 virus presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody therapies and vaccine-induced sera. Decoy nanoparticles-cell-mimicking particles that bind and inhibit virions-are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, we lack quantitative understanding as to how design features impact performance of these therapeutics. To address this gap, here we perform a systematic, comparative evaluation of various biologically-derived nanoscale vesicles, which may be particularly well-suited to sustained or repeated administration in the clinic due to low toxicity, and investigate their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade monoclonal antibodies and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

20.
Cell Mol Bioeng ; 14(5): 459-469, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34777604

RESUMEN

INTRODUCTION: The design of sensors that can detect biological ions in situ remains challenging. While many fluorescent indicators exist that can provide a fast, easy readout, they are often nonspecific, particularly to ions with similar charge states. To address this issue, we developed a vesicle-based sensor that harnesses membrane channels to gate access of potassium (K+) ions to an encapsulated fluorescent indicator. METHODS: We assembled phospholipid vesicles that incorporated valinomycin, a K+ specific membrane transporter, and that encapsulated benzofuran isophthalate (PBFI), a K+ sensitive dye that nonspecifically fluoresces in the presence of other ions, like sodium (Na+). The specificity, kinetics, and reversibility of encapsulated PBFI fluorescence was determined in a plate reader and fluorimeter. The sensors were then added to E. coli bacterial cultures to evaluate K+ levels in media as a function of cell density. RESULTS: Vesicle sensors significantly improved specificity of K+ detection in the presence of a competing monovalent ion, sodium (Na+), and a divalent cation, calcium (Ca2+), relative to controls where the dye was free in solution. The sensor was able to report both increases and decreases in K+ concentration. Finally, we observed our vesicle sensors could detect changes in K+ concentration in bacterial cultures. CONCLUSION: Our data present a new platform for extracellular ion detection that harnesses ion-specific membrane transporters to improve the specificity of ion detection. By changing the membrane transporter and encapsulated sensor, our approach should be broadly useful for designing biological sensors that detect an array of biological analytes in traditionally hard-to-monitor environments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00688-7.

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