More Like this

1. Facile Formation of Giant Elastin-like Polypeptide Vesicles as Synthetic Cells

   https://doi.org/10.26434/chemrxiv.13718194.v1  

   Bineet Sharma, Yutao Ma ( February 2021 , ChemRxiv)

   null (Ed.)

   Creating a suitable compartment for synthetic cells has led the exploration of different cell chassis materials from phospholipids to polymer to protein-polymer conjugates. Currently, the majority of cell-like compartments are made of lipid molecules as the resulting membrane resembles that of a natural cell. However, cell-sized lipid vesicles are prone to physical and chemical stresses and can be unstable in hosting biochemical reactions within. Recently, peptide vesicles that are more robust and stable were developed as a new chassis material for synthetic cells. Here we demonstrate the facile and robust generation of giant peptide vesicles made of elastin-like polypeptides (ELPs) by using an emulsion transfer method. We show that these peptide vesicles can stably encapsulate molecules and can host cell-free expression reactions. We also demonstrate membrane incorporation of another amphiphilic ELP into existing peptide vesicles. Since ELPs are genetically encoded, the approaches presented here provide exciting opportunities to engineer synthetic cell membranes. 

   more » « less
2. Computational Design of Self-Assembling Peptide Chassis Materials for Synthetic Cells

   https://doi.org/10.1039/D2ME00169A  

   Ma, Yutao ; Kapoor, Rohan ; Sharma, Bineet ; Liu, Allen ; Ferguson, Andrew L ( January 2022 , Molecular Systems Design & Engineering)

   Giant lipid vesicles have been used extensively as a synthetic cell model to recapitulate various life-like processes, including in vitro protein synthesis, DNA replication, and cytoskeleton organization. Cell-sized lipid vesicles are mechanically fragile in nature and prone to rupture due to osmotic stress, which limits their usability. Recently, peptide vesicles have been introduced as an alternative chassis material for synthetic cells that are more robust and stable than lipid vesicles, and can withstand harsh conditions including pH, thermal, and osmotic variations. In this work, we combine coarse-grained molecular simulation, enhanced sampling free energy calculations, Gaussian process regression, and Bayesian optimization to construct an active learning screening for diblock amphiphilic elastin-like polypeptides capable of forming thermodynamically stable vesicular structures suitable for the self-assembly of synthetic peptide vesicles. Our computational screen identifies a number of promising sequences that form peptidic vesicles with high thermodynamic stabilities relative to isolated peptides in bulk solvent on the order of 10-15 k B T per amino acid residue. 

   more » « less
3. In search of a novel chassis material for synthetic cells: emergence of synthetic peptide compartment

   https://doi.org/10.1039/d0sm01644f  

   Sharma, Bineet ; Ma, Yutao ; Ferguson, Andrew L. ; Liu, Allen P. ( December 2020 , Soft Matter)

   null (Ed.)

   Giant lipid vesicles have been used extensively as a synthetic cell model to recapitulate various life-like processes, including in vitro protein synthesis, DNA replication, and cytoskeleton organization. Cell-sized lipid vesicles are mechanically fragile in nature and prone to rupture due to osmotic stress, which limits their usability. Recently, peptide vesicles have been introduced as a synthetic cell model that would potentially overcome the aforementioned limitations. Peptide vesicles are robust, reasonably more stable than lipid vesicles and can withstand harsh conditions including pH, thermal, and osmotic variations. This mini-review summarizes the current state-of-the-art in the design, engineering, and realization of peptide-based chassis materials, including both experimental and computational work. We present an outlook for simulation-aided and data-driven design and experimental realization of engineered and multifunctional synthetic cells. 

   more » « less
4. Hydrophobe Containing Polypeptoids Complex with Lipids and Induce Fusogenesis of Lipid Vesicles

   Marzhana Omarova, Yueheng Zhang ( January 2021 , Journal of physical chemistry)

   The hydrophobic effect of alkyl group insertion into phospholipid bilayers is exploited in modifying and modulating vesicle structure. We show that amphiphilic polypeptoids (peptide mimics) with n-decyl side chains, which we term as hydrophobe-containing polypeptoids (HCPs), can insert the alkyl hydrophobes into the membrane bilayer of phospholipid-based vesicles. Such insertion leads to disruption of the liposomes and the formation of HCP–lipid complexes that are colloidally stable in aqueous solution. Interestingly, when these complexes are added to fresh liposomes, remnant uncomplexed hydrophobes (the n-decyl groups) bridge liposomes and fuse them. The fusion leads to the engulfing of liposomes and the formation of multilayered vesicles. The morphology of the liposome system can be changed from stopping fusion and forming clustered vesicles to the continued formation of multilayered liposomes simply by controlling the amount of the HCP–lipid complex added. The entire procedure occurs in aqueous systems without the addition of any other solvents. There are several implications to these observations including the biological relevance of mimicking fusogenic proteins such as the SNARE proteins and the development of new drug delivery technologies to impact delivery to cell organelles. 

   more » « less
5. Production of Class II MHC Proteins in Lentiviral Vector‐Transduced HEK‐293T Cells for Tetramer Staining Reagents

   https://doi.org/10.1002/cpz1.36  

   Willis, Richard A. ; Ramachandiran, Vasanthi ; Shires, John C. ; Bai, Ge ; Jeter, Kelly ; Bell, Donielle L. ; Han, Lixia ; Kazarian, Tamara ; Ugwu, Kyla C. ; Laur, Oskar ; et al ( February 2021 , Current Protocols)

   Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed inE. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.

   This article was corrected on 19 July 2022. See the end of the full text for details.

   Basic Protocol 1: Lentivirus production and expression line creation

   Support Protocol 1: Six‐well assay for estimation of production cell line yield

   Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

   Basic Protocol 2: Cultures for production of Class II MHC proteins

   Basic Protocol 3: Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatography

   Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

   Support Protocol 3: Protein concentration measurements and adjustments

   Support Protocol 4: Polishing purification by anion‐exchange chromatography

   Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

   Basic Protocol 4: Peptide exchange

   Basic Protocol 5: Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry

   Alternate Protocol 2: Native isoelectric focusing to validate MHC‐II peptide loading

   Basic Protocol 6: Multimerization

   Basic Protocol 7: Staining cells with Class II MHC tetramers

    

   more » « less