This content will become publicly available on December 1, 2022
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- Nature Communications
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- National Science Foundation
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Abstract Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic N-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines.
Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane–membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane–membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell-free expression (CFE) system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane–membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful where cell-cell contacts and communication are recreated in a controlled manner using minimal components.
Cell-like hybrids from natural and synthetic amphiphiles provide a platform to engineer functions of synthetic cells and protocells. Cell membranes and vesicles prepared from human cell membranes are relatively unstable in vitro and therefore are difficult to study. The thicknesses of biological membranes and vesicles self-assembled from amphiphilic Janus dendrimers, known as dendrimersomes, are comparable. This feature facilitated the coassembly of functional cell-like hybrid vesicles from giant dendrimersomes and bacterial membrane vesicles generated from the very stable bacterial
Escherichia colicell after enzymatic degradation of its outer membrane. Human cells are fragile and require only mild centrifugation to be dismantled and subsequently reconstituted into vesicles. Here we report the coassembly of human membrane vesicles with dendrimersomes. The resulting giant hybrid vesicles containing human cell membranes, their components, and Janus dendrimers are stable for at least 1 y. To demonstrate the utility of cell-like hybrid vesicles, hybrids from dendrimersomes and bacterial membrane vesicles containing YadA, a bacterial adhesin protein, were prepared. The latter cell-like hybrids were recognized by human cells, allowing for adhesion and entry of the hybrid bacterial vesicles into human cells in vitro.
Sedimentary rocks host a vast reservoir of organic carbon, such as 2-methylhopane biomarkers, whose evolutionary significance we poorly understand. Our ability to interpret this molecular fossil record is constrained by ignorance of the function of their molecular antecedents. To gain insight into the meaning of 2-methylhopanes, we quantified the dominant (des)methylated hopanoid species in the membranes of the model hopanoid-producing bacterium Rhodopseudomonas palustris TIE-1. Fluorescence polarization studies of small unilamellar vesicles revealed that hopanoid 2-methylation specifically renders native bacterial membranes more rigid at concentrations that are relevant in vivo. That hopanoids differentially modify native membrane rigidity as a function of their methylation state indicates that methylation itself promotes fitness under stress. Moreover, knowing the in vivo (2Me)-hopanoid concentration range in different cell membranes, and appreciating that (2Me)-hopanoids' biophysical effects are tuned by the lipid environment, permits the design of more relevant in vitro experiments to study their physiological functions.
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.