Programming protein phase-separation employing a modular library of intrinsically disordered precision block copolymer-like proteins creating dynamic cytoplasmatic compartmentalization.

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.

High-throughput screening of optimal process conditions using model predictive control.

Modern biotechnological laboratories are equipped with advanced parallel mini-bioreactor facilities that can perform sophisticated cultivation strategies (e.g., fed-batch or continuous) and generate significant amounts of measurement data.

Purification of a Hydrophobic Elastin-Like Protein Toward Scale-Suitable Production of Biomaterials.

Elastin-like proteins (ELPs) are polypeptides with potential applications as renewable bio-based high-performance polymers, which undergo a stimulus-responsive reversible phase transition. The ELP investigated in this manuscript-ELP[V2Y-45]-promises fascinating mechanical properties in biomaterial applications. Purification process scalability and purification performance are important factors for the evaluation of potential industrial-scale production of ELPs.

Dynamic Structural Changes and Thermodynamics in Phase Separation Processes of an Intrinsically Disordered-Ordered Protein Model.

Elastin-like proteins (ELPs) are biologically important proteins and models for intrinsically disordered proteins (IDPs) and dynamic structural transitions associated with coacervates and liquid-liquid phase transitions. However, the conformational status below and above coacervation temperature and its role in the phase separation process is still elusive. Employing matrix least-squares global Boltzmann fitting of the circular dichroism spectra of the ELPs (VPGVG) , (VPGVG) , and (VPGVG) , we found that coacervation occurs sharply when a certain number of repeat units has acquired β-turn conformation (in our sequence setting a threshold of approx.

Towards a Continuous Manufacturing Process of Protein-Loaded Polymeric Nanoparticle Powders.

To develop a scalable and efficient process suitable for the continuous manufacturing of poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing ovalbumin as the model protein. PLGA nanoparticles were prepared using a double emulsification spray-drying method. Emulsions were prepared using a focused ultrasound transducer equipped with a flow cell.

Sortase-A mediated chemoenzymatic lipidation of single-domain antibodies for cell membrane engineering.

Purpose: Membrane engineering has versatile applications in adoptive cell therapies, immune therapy or drug delivery. Incorporation of lipidated antibody-derived ligands into cells may enforce supraphysiological cell interactions that offer new therapeutic approaches. A challenge is the defined synthesis of lipidated ligands that effectively interact with such membranes.

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.

Adjustable Bioorthogonal Conjugation Platform for Protein Studies in Live Cells Based on Artificial Compartments.

The investigation of complex biological processes often requires defined multiple bioconjugation and positioning of functional entities on 3D structures. Prominent examples include spatially defined protein complexes in nature, facilitating efficient biocatalysis of multistep reactions. Mimicking natural strategies, synthetic scaffolds should comprise bioorthogonal conjugation reactions and allow for absolute stoichiometric quantification as well as facile scalability through scaffold reproduction.

Prebiotic Protocell Model Based on Dynamic Protein Membranes Accommodating Anabolic Reactions.

The nature of the first prebiotic compartments and their possible minimal molecular composition is of great importance in the origin of life scenarios. Current protocell model membranes are proposed to be lipid-based. This paradigm has several shortcomings such as limited membrane stability of monoacyl lipid-based membranes (e.

Minimalist Protocell Design: A Molecular System Based Solely on Proteins that Form Dynamic Vesicular Membranes Embedding Enzymatic Functions.

Life in its molecular context is characterized by the challenge of orchestrating structure, energy and information processes through compartmentalization and chemical transformations amenable to mimicry of protocell models. Here we present an alternative protocell model incorporating dynamic membranes based on amphiphilic elastin-like proteins (ELPs) rather than phospholipids. For the first time we demonstrate the feasibility of combining vesicular membrane formation and biocatalytic activity with molecular entities of a single class: proteins.

Self-Assembly Toolbox of Tailored Supramolecular Architectures Based on an Amphiphilic Protein Library.

The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self-assembly of supramolecular structures with desired shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self-assembly of tailored amphiphilic elastin-like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced.

Sortagged anti-EGFR immunoliposomes exhibit increased cytotoxicity on target cells.

Purpose: Conventional chemotherapy is associated with therapy-limiting side effects, which might be alleviated by targeted chemotherapeutics such as immunoliposomes. The targeting ligands of immunoliposomes are commonly attached by unspecific chemical conjugation, bearing risk of structural heterogeneity and therewith related biological consequences. Chemoenzymatic methods may mitigate such risks through site-specific conjugation.

Sortagging of liposomes with a murine CD11b-specific VHH increases in vitro and in vivo targeting specificity of myeloid cells.

The therapeutic index of drugs can be increased via drug encapsulation in actively targeted, meaning ligand modified drug delivery systems. The manufacturing of such targeted drug delivery systems, in particular the conjugation between drug carrier and ligand, can be done by enzymatic conjugation methods, exploiting the site-specific, bioorthogonal nature of these reactions. The use of such enzymes like Sortase-A transpeptidase requires efficient purification methods, as residuals of the enzyme may be responsible for immunogenic potential and drug product instabilities.

Sortaggable liposomes: Evaluation of reaction conditions for single-domain antibody conjugation by Sortase-A and targeting of CD11b myeloid cells.

Active targeting with ligand coated liposomal drug delivery systems is a means to increase the therapeutic index of drugs. Stable ligand coating requires bilayer anchorage of the commonly proteinaceous ligands and hence a conjugation of lipid structures towards amino acids. This often leads to heterogeneous reaction products especially when chemical coupling methods are employed.

Pentaglycine lipid derivates - rp-HPLC analytics for bioorthogonal anchor molecules in targeted, multiple-composite liposomal drug delivery systems.

The quantification of lipids and assessment of lipid composition is an indispensable step during the pharmaceutical development of novel lipid based drug delivery systems such as liposomes. Broad excipient screenings of such formulations raise the need for versatile analytical methods. Even more demanding complexity is generated by introduction of targeted systems requiring functionalized lipids.

[Tissue engineering of adipose tissue with biodegradable biomaterials for soft tissue reconstruction].

Soft tissue defects resulting from injuries, tumor resection, congenital anomalies or chronic wounds pose a great challenge to reconstructive surgery. The current gold standard in therapy of such defects is the tissue transplantation in terms of free or local flaps. Unfortunately, donor site morbidity remains a considerable risk of flap surgery.

Transient apoptosis inhibition in donor stem cells improves hematopoietic stem cell transplantation.

During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity.

Automated detection of protein unfolding events in atomic force microscopy force curves.

Atomic force microscopy is not only a high-resolution imaging device but also a mechanical machine, which can be used either to indent or stretch (soft) biomaterials. Due to the statistical nature of such materials (i.e.

Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids.

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells.

Hemolysin coregulated protein 1 as a molecular gluing unit for the assembly of nanoparticle hybrid structures.

Hybrid nanoparticle (NP) structures containing organic building units such as polymers, peptides, DNA and proteins have great potential in biosensor and electronic applications. The nearly free modification of the polymer chain, the variation of the protein and DNA sequence and the implementation of functional moieties provide a great platform to create inorganic structures of different morphology, resulting in different optical and magnetic properties. Nevertheless, the design and modification of a protein structure with functional groups or sequences for the assembly of biohybrid materials is not trivial.

Focused Ultrasound as a Scalable and Contact-Free Method to Manufacture Protein-Loaded PLGA Nanoparticles.

Purpose: Although nanomaterials are under investigation for a very broad range of medical applications, only a small fraction of these are already commercialized or in clinical development. A major challenge for the translation of nanomedicines into the clinic is the missing scalability of the available lab scale preparation methods and, ultimately, non-identical samples during early and late research.

Methods: Protein-loaded PLGA nanoparticles using focused ultrasound in an emulsion solvent diffusion method were prepared in different batch sizes to evaluate achievable mean size, protein loading, and yield.

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures.

The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP).

Reversible biomechano-responsive surface based on green fluorescent protein genetically modified with unnatural amino acids.

GFP has been genetically modified at two specific positions of its molecular architecture. These modifications allow its covalent attachment onto PEG brushes grafted on functionalized silicone surfaces. The stretching of this material leads to a reversible decrease of the fluorescence intensity due to stretch-induced forces applying on GFP molecules.

Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments.

Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles.

Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid-materials.

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.