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The control of supramolecular complexes in living systems at the molecular level is an important goal in life-sciences. Spatiotemporal organization of molecular distribution & flow of such complexes are essential physicochemical processes in living cells and important for pharmaceutical processes. Membraneless organelles (MO) found in eukaryotic cells, formed by liquid-liquid phase-separation (LLPS) of intrinsically disordered proteins (IDPs) control and adjust intracellular organization. Artificially designed compartments based on LLPS open up a novel pathway to control chemical flux and partition in vitro and in vivo. We designed a library of chemically precisely defined block copolymer-like proteins based on elastin-like proteins (ELPs) with defined charge distribution and type, as well as polar and hydrophobic block domains. This enables the programmability of physicochemical properties and to control adjustable LLPS in vivo attaining control over intracellular partitioning and flux as role model for in vitro and in vivo applications. Tailor-made ELP-like block copolymer proteins exhibiting IDP-behavior enable LLPS formation in vitro and in vivo allowing the assembly of membrane-based and membraneless superstructures via protein phase-separation in E. coli. Subsequently, we demonstrate the responsiveness of protein phase-separated spaces (PPSSs) to environmental physicochemical triggers and their selective, charge-dependent and switchable interaction with DNA or extrinsic and intrinsic molecules enabling their selective shuttling across semipermeable phase boundaries including (cell)membranes. This paves the road for adjustable artificial PPSS-based storage and reaction spaces and the specific transport across phase boundaries for applications in pharmacy and synthetic biology. • An amphiphilic protein (aELP) library allows for adjustable compartments in vivo. • Extrinsic stimuli modulate protein-phase separated spaces (PPSS) in E. coli. • PPSS formed by charged aELPs direct DNA localization. • Charged aELP forming PPSS can control molecular flux of molecules in vivo. • Responsive PPSS can be used in pharmaceutical applications and synthetic biology. [ABSTRACT FROM AUTHOR]

The access to defined protein-based material systems is a major challenge in bionanotechnology and regenerative medicine. Exact control over sequence composition and modification is an important requirement for the intentional design of structure and function. Herein structural- and matrix proteins provide a great potential, but their large repetitive sequences pose a major challenge in their assembly. Here we introduce an integrative "one-vector-toolbox-platform" (OVTP) approach which is fast, efficient and reliable. The OVTP allows for the assembly, multimerization, intentional arrangement and direct translation of defined molecular DNA-tecton libraries, in combination with the selective functionalization of the yielded protein-tecton libraries. The diversity of the generated tectons ranges from elastine-, resilin, silk- to epitope sequence elements. OVTP comprises the expandability of modular biohybrid-materials via the assembly of defined multi-block domain genes and genetically encoded unnatural amino acids (UAA) for site-selective chemical modification. Thus, allowing for the modular combination of the protein-tecton library components and their functional expansion with chemical libraries via UAA functional groups with bioorthogonal reactivity. OVTP enables access to multitudes of defined protein-based biohybrid-materials for self-assembled superstructures such as nanoreactors and nanobiomaterials, e.g. for approaches in biotechnology and individualized regenerative medicine. [ABSTRACT FROM AUTHOR]