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Abstract Synthetic biology has enabled the development of new strategies for creating artificial cells that can sense and respond to external stimuli. This study introduces the bottom-up construction of globular protein vesicles (GPVs) that incorporate elastin-like peptide (ELP) bolaamphiphiles as transmembrane components. To enable this strategy, we devised a Golden Gate-based cloning strategy to streamline the design, expression, and purification of ELP bolaamphiphiles. Three ELP bolamphiphiles with varying structural complexity were developed, incorporating fluorescent proteins to facilitate visualization and characterization. The self-assembly of these bolaamphiphiles into GPVs was optimized by varying the molar ratios of recombinant building blocks. Structural characterization confirmed vesicle formation, dynamic light scattering analysis revealed size distributions dependent on modular complexity, and atomic force microscopy demonstrated that the vesicles exhibited MPa-range Young’s moduli, indicative of high mechanical robustness. Our findings demonstrate that multifunctional ELP bolaamphiphiles can be incorporated into GPVs, enabling modular vesicle engineering. This work provides a foundation for designing synthetic cells with customizable bi-functionalities and modularity, advancing compartmentalized systems. 

This study investigates the phase transition of globular protein vesicles – a model for protein-constructed artificial cells and organelles – under macromolecular crowding conditions in biomimetic environments. 

Synthetic vesicles have gained considerable popularity in recent years for numerous biological and medical applications. Among the various types of synthetic vesicles, the utilization of polypeptides and/or proteins as fundamental constituents has garnered significant interest for vesicle construction owing to the unique bio-functionalities inherent in rationally designed amino acid sequences. Especially the incorporation of functional proteins onto the vesicle surface facilitates a wide range of advanced biological applications that are not easily attainable with traditional building blocks, such as lipids and polymers. The main goal of this review is to provide a comprehensive overview of the latest advancements in polypeptide/protein vesicles. Moreover, this review encompasses the rational design and engineering strategies employed in the creation of polypeptide/protein vesicles, including the synthesis of building blocks, the modulation of their self-assembly, as well as their diverse applications. Furthermore, this work includes an in-depth discussion of the key challenges and opportunities associated with polypeptide/protein vesicles, providing valuable insights for future research. By offering an up-to-date review of this burgeoning field of polypeptide/protein vesicle research, this review will shed light on the potential applications of these biomaterials. 

In this study, we investigate the changes in the permeability of the recombinant fusion protein vesicles with different membrane structures as a function of solution temperature. The protein vesicles are self-assembled from recombinant fusion protein complexes composed of an mCherry fused with a glutamic acid-rich leucine zipper and a counter arginine-rich leucine zipper fused with an elastin-like polypeptide (ELP). We have found that the molecular weight cut-off (MWCO) of the protein vesicle membranes varies inversely with solution temperature by monitoring the transport of fluorescent-tagged dextran dyes with different molecular weights. The temperature-responsiveness of the protein vesicle membranes is obtained from the lower critical solution temperature behavior of ELP in the protein building blocks. Consequently, the unique vesicle membrane structures with different single-layered and double-layered ELP organizations impact the sensitivity of the permeability responses of the protein vesicles. Single-layered protein vesicles with the ELP domains facing the interior show more drastic permeability changes as a function of temperature than double-layered protein vesicles in which ELP blocks are buried inside the membranes. This work about the temperature-responsive membrane permeability of unique protein vesicles will provide design guidelines for new biomaterials and their applications, such as drug delivery and synthetic protocell development.