The 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 Ni 2+-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.
The Dutch Ministry of Education, Culture, and Science (Gravitation Program 024.001.035) and the ERC Advanced Grant (Artisym 694120) are acknowledged for funding. Joost L. J. van Dongen and Iris A. Leijten - van de Gevel are thanked for the acquisition of the LC/MS Q-TOF data.
|Dutch Ministry of Education, Culture, and Science
|Horizon 2020 Framework Programme
|European Research Council