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. 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.

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. These systems require not only optimal experimental designs that find the best conditions in very large design spaces, but also algorithms that manage to operate a large number of different cultivations in parallel within a well-defined and tightly constrained operating regime. Existing advanced process control algorithms have to be tailored to tackle the specific issues of such facilities such as: a very complex biological system, constant changes in the metabolic activity and phenotypes, shifts of pH and/or temperature, and metabolic switches, to name a few. In this study we implement a model predictive control (MPC) framework to demonstrate: (1) the challenges in terms of mathematical model structure, state, and parameter estimation, and optimization under highly nonlinear and stiff dynamics in biological systems, (2) the adaptations required to enable the application of MPC in high throughput bioprocess development, and (3) the added value of MPC implementations when operating parallel mini-bioreactors aiming to maximize the biomass concentration while coping with hard constrains on the dissolved oxygen tension profile.

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. Salt-induced precipitation, inverse transition cycling (ITC), and immobilized metal ion affinity chromatography (IMAC) were assessed as purification protocols for a polyhistidine-tagged hydrophobic ELP showing low-temperature transition behavior. IMAC achieved a purity of 86% and the lowest nucleic acid contamination of all processes. Metal ion leakage did not propagate chemical modifications and could be successfully removed through size-exclusion chromatography. The simplest approach using a high-salt precipitation resulted in a 60% higher target molecule yield compared to both other approaches, with the drawback of a lower purity of 60% and higher nucleic acid contamination. An additional ITC purification led to the highest purity of 88% and high nucleic acid removal. However, expensive temperature-dependent centrifugation steps are required and aggregation effects even at low temperatures have to be considered for the investigated ELP. Therefore, ITC and IMAC are promising downstream processes for biomedical applications with scale-dependent economical costs to be considered, while salt-induced precipitation may be a fast and simple alternative for large-scale bio-based polymer production.

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)20 , (VPGVG)40 , and (VPGVG)60 , we found that coacervation occurs sharply when a certain number of repeat units has acquired ß-turn conformation (in our sequence setting a threshold of approx. 20 repeat units). The character of the differential scattering of the coacervate suspensions indicated that this fraction of ß-turn structure is still retained after polypeptide assembly. Such conformational thresholds may also have a role in other protein assembly processes with implications for the design of protein-based smart materials.

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. Either poly(vinyl alcohol) (PVA) or poloxamer 407 (P-407) was used as a stabilizer. Aliquots of the emulsions were blended with different matrix excipients and spray dried, and the yield and size of the resuspended nanoparticles was determined and compared against solvent displacement. Nanoparticle sizes of spray-dried PLGA/PVA emulsions were independent of the matrix excipient and comparable with sizes from the solvent displacement method. The yield of the resuspended nanoparticles was highest for emulsions containing trehalose and leucine (79%). Spray drying of PLGA/P-407 emulsions led to agglomerated nanoparticles independent of the matrix excipient. PLGA/P-407 nanoparticles pre-formed by solvent displacement could be spray dried with limited agglomeration when PVA was added as an additional stabilizer. A comparably high and economically interesting nanoparticle yield could be achieved with a process suitable for continuous manufacturing. Further studies are needed to understand the robustness of a continuous process at commercial scale.

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. METHODS: Sortase-A was used to attach a PEGylated, dimyristyl lipid-anchor on single-domain antibodies (VHH). The membrane insertion was investigated on liposomal bilayers, myeloid-derived suppressor cells (MDSC) and T cells. RESULTS: The lipidated VHHs remodeled liposomal as well as cellular membranes. The VHH carrying liposomes were successfully targeted towards antigen-positive cells. MDSC and T cells were both modified with lipidated VHHs as detected with an FITC-anti-llama antibody. T cells that carried an anti-CD11b VHH showed cellular association in vitro with CD11b+Gr-1+ MDSC in a two-dimensional magnetic activated cell sorting / flow-cytometry assay. CONCLUSION: The applied combination of chemoenzymatic ligation, PEGylated lipid anchors and single-domain antibodies delivers water-soluble and chemically defined lipidated ligands, which readily associate with liposomal and cellular membranes. This enables liposomal drug targeting and artificial cell-cell interactions. Hence, the presented concept for lipidation of single-domain antibodies is promising for further application in the field of drug delivery or cell-based therapies.

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. Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

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

The investigation of complex biological processes in vivo 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. Existing in vivo scaffolding strategies often lack covalent conjugations on geometrically confined scaffolds or precise quantitative characterization. Addressing these shortcomings, we present a bioorthogonal dual conjugation platform based on genetically encoded artificial compartments in vivo, comprising two distinct genetically encoded covalent conjugation reactions and their precise stoichiometric quantification. The SpyTag/SpyCatcher (ST/SC) bioconjugation and the controllable strain-promoted azide-alkyne cycloaddition (SPAAC) were implemented on self-assembled protein membrane-based compartments (PMBCs). The SPAAC reaction yield was quantified to be 23% ± 3% and a ST/SC surface conjugation yield of 82% ± 9% was observed, while verifying the compatibility of both chemical reactions as well as enhanced proteolytic stability. Using tandem mass spectrometry, absolute concentrations of the proteinaceous reactants were calculated to be 0.11 ± 0.05 attomol/cell for PMBC surface-tethered mCherry-ST-His and 0.22 ± 0.09 attomol/cell for PMBC-constituting pAzF-SC-E20F20-His. The established in vivo conjugation platform enables quantifiable protein-protein interaction studies on geometrically defined scaffolds and paves the road to investigate effects of scaffold-tethering on enzyme activity.

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.g., fatty acids), missing pathways to synthesize protocell membrane components (e.g., phospholipids) under early earth conditions, and the requirement for different classes of molecules for the formation of compartments and the catalysis of reactions. Amino acids on the other hand are known to arise and persist with remarkable abundance under early earth conditions since the fundamental Miller-Urey experiments. They were also postulated early to form protocellular structures, for example, proteinoid capsules. Here, we present a protocell model constituted by membranes assembled from amphiphilic proteins based on prebiotic amino acids. Self-assembled dynamic protein membrane-based compartments (PMBCs) are impressively stable and compatible with prevalent cellular membrane constituents forming protein-only or protein-lipid hybrid membranes. They can embed processes essential for extant living cells, such as enclosure of molecules, membrane fusion, phase separation, and complex biosynthetic elements from modern cells demonstrating "upward" compatibility. Our findings suggest that prebiotic PMBCs represent a new type of protocell as a possible ancestor of current lipid-based cells. The presented prebiotic PMBC model can be used to design artificial cells, important for the study of structural, catalytic, and evolutionary pathways related to the emergence of life.

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. The described assembly protocols efficiently yield protein membrane-based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.

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. The presented self-assembled protein-membrane-based compartments (PMBCs) accommodate either an anabolic reaction, based on free DNA ligase as an example of information transformation processes, or a catabolic process. We present a catabolic process based on a single molecular entity combining an amphiphilic protein with tobacco etch virus (TEV) protease as part of the enclosure of a reaction space and facilitating selective catalytic transformations. Combining compartmentalization and biocatalytic activity by utilizing an amphiphilic molecular building block with and without enzyme functionalization enables new strategies in bottom-up synthetic biology, regenerative medicine, pharmaceutical science and biotechnology.

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. METHODS: The formulation parameters for pentaglycine-modified, doxorubicin-loaded liposomes and the reaction conditions for a site-specific, Sortase-A mediated conjugation with monoclonal antibodies were thoroughly evaluated. The cytotoxicity of such sortagged, epidermal growth factor receptor (EGFR)-specific immunoliposomes was tested on human breast cancer cells. RESULTS: Sortaggable liposomes with a defined size (140 nm, PDI < 0.25) and high encapsulation efficiency (>90%) were obtained after manufacturing optimization. A ratio of 1.0-2.5 µM mAb/100 µM pentaglycine yielded stable dispersions and circumvented carrier precipitation during ligand grafting. The cytotoxicity on EGFR+ MDA-MB-468 was up to threefold higher for EGFR-specific immunoliposomes than for the nontargeted controls. CONCLUSIONS: Sortase-A is suitable to generate immunoliposomes with a site-specific ligand-carrier linkage and hence improves chemical homogeneity of targeted therapeutics. However, the sweet spot for manufacturability utilizing mAbs with two Sortase-A recognition sites is narrow, making mono-reactive binders such as scFvs or Fab's preferable for a further development. Despite this, the immunoliposomes demonstrated a targeted delivery of doxorubicin, indicating the potential to increase the therapeutic window during the treatment of EGFR+ tumors.

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. These instabilities may be based on the enzymatic reverse reaction, meaning here a cleavage between ligand and drug carrier. In the presented work, two differently PEGylated formulations were modified with variable fragments of camelid heavy chain-only antibodies (VHH) via Sortase-A, purified by different methodologies and tested for ligand cleavage upon storage. Strongly PEGylated liposomes (PEGhigh-LS) were found to retain higher amounts of Sortase-A than lowly PEGylated ones (PEGlow-LS) after dialysis purification. Surprisingly, this did not correlate with ligand stability during storage. PEGhigh-LS were less prone for degradation, compared to PEGlow-LS, which showed a ligand cleavage of 20% after an 8 weeks storage at 2-8 °C. Nonetheless, overall degradation could be minimized by an additional affinity bead purification procedure. Liposomes modified with a CD11b-specific VHH were tested for their in vitro and in vivo targeting ability towards CD11b+ cells. Specific targeting of CD11b was achieved in vitro and in vivo on various cell types. PEGylation decreased the targeting effect in vitro, however no differences between PEGhigh or PEGlow formulations were observed in vivo. The obtained results underline the need for a thorough characterization of novel conjugation strategies as well as an early in vivo characterization of such targeted drug delivery systems.

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. Chemoenzymatic Sortase-A mediated transpeptidation (sortagging) is a useful tool to avoid this protein heterogeneity through its site-specific, bioorthogonal ligation mechanism. Manufacturing of such sortaggable, pentaglycine modified liposomes was developed by adaption of a scalable solvent injection technique. The pentaglycine liposomes were prepared with different degrees of PEGylation and steric accessibility of the pentaglycine motif. Comparable hydrodynamic diameters (146-188 nm) of the different formulations were obtained after a flow rate screening. The sortagging reactivity of a single-domain antibody (VHH) towards the pentaglycine liposomes was strongly dependent on the steric accessibility of the pentaglycine nucleophile. Adjusting the pentaglycine to ligand ratio improved conversion rates up to 80%. The liposome bound VHH was accessible for its soluble antigen as shown by a chromatography-based binding assay. Mono- and granulocytes could be selectively targeted in vitro by conjugation of BMX1, a VHH directed towards human myeloid cell surface marker CD11b. Confocal microscopy revealed intracellular localization of the targeted liposomes. The developability of those pentaglycine liposomes as well as their proof of principle for targeted drug delivery shows their potential for further investigation, for example as delivery platform for diagnostics or drugs into the tumor microenvironment.

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. We addressed this demand by developing an rp-HPLC based analytical method with evaporative light scattering detection (ELSD) for the simultaneous analysis of commonly used phosphatidylcholines, cholesterol and bilayer surface-modifying cationic, anionic or PEGylated lipids, which can be analyzed in combination with novel pentaglycine lipids suitable as targeting ligand anchor. The method was validated for specificity, precision, accuracy and sample stability. We monitor the continuous and scalable manufacturing of two pentaglycine-modified liposomal formulations and track the modification of these drug delivery systems with a single-domain antibody utilizing bioorthogonal Sortase-A technology. Both the presented analytical and preparative techniques can help to improve the quality control and to accelerate the pharmaceutical development of such targeted drug delivery systems.

[Tissue engineering of adipose tissue with biodegradable biomaterials for soft tissue reconstruction]. / Tissue Engineering von Fettgewebe mittels bioabbaubaren Biomaterialien zur Weichteildefektdeckung.

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. Therefore, tissue engineering of autologous vascularized long term stable adipose tissue constructs could enrich the therapeutic possibilities of soft tissue defects. De novo adipose tissue growing requires fundamental knowledge about this kind of tissue and its synthesis, closely linked to angiogenesis. Bioresorbable biomaterials (scaffolds) are of crucial importance for adipose tissue engineering. Simulation or replacement of extracellular matrix for tissue growth by scaffold application requires a profound understanding of cell-matrix interactions. A proper biomaterial should be capable of supporting cell adherence, proliferation and differentiation. Important features are biocompatibility and resorption without toxic metabolites. In this review, various scaffold materials are discussed and novel achievements are presented. Persisting problems of de novo adipose tissue formation are high resorption rates and small tissue volumes of adipose constructs. Adipose tissue formation in a tissue engineering chamber is an additional possibility for in vivo tissue engineering. Recent studies proof that long term stable de novo adipose tissue formation of clinically relevant tissue volumes is possible. This method, in our opinion, has the potential to improve therapeutic strategies of soft tissue defects significantly.

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. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.

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. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

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., hydrogels or polymers) hundreds of force-distance curves are required to describe their mechanical properties. In this manuscript, we present an automated system for polymer unfolding detection based on continuous wavelet analysis. We have tested the automated program on elastin, which is an important protein that provides elasticity to tissues and organs. Our results show that elastin changes its mechanical behavior in the presence of electrolytes. In particular, we show that NaCl has a different effect on the contour length than CaCl2 for similar unfolding forces. In addition, we provide the program in the supporting information for the researches facing such kind of problem.

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. This is mainly due to the sensitivity of its secondary, tertiary and quaternary structure to the changes in the interaction (e.g., hydrophobic, hydrophilic, electrostatic, chemical groups) between the protein subunits and the inorganic material. Here, we use hemolysin coregulated protein 1 (Hcp1) from Pseudomonas aeruginosa as a building and gluing unit for the formation of biohybrid structures by implementing cysteine anchoring points at defined positions on the protein rim (Hcp1_cys3). We successfully apply the Hcp1_cys3 gluing unit for the assembly of often linear, hybrid structures of plasmonic gold (Au NP), magnetite (Fe3O4 NP), and cobalt ferrite nanoparticles (CoFe2O4 NP). Furthermore, the assembly of Au NPs into linear structures using Hcp1_cys3 is investigated by UV-vis spectroscopy, TEM and cryo-TEM. One key parameter for the formation of Au NP assembly is the specific ionic strength in the mixture. The resulting network-like structure of Au NPs is characterized by Raman spectroscopy, showing surface-enhanced Raman scattering (SERS) by a factor of 8·10(4) and a stable secondary structure of the Hcp1_cys3 unit. In order to prove the catalytic performance of the gold hybrid structures, they are used as a catalyst in the reduction reaction of 4-nitrophenol showing similar catalytic activity as the pure Au NPs. To further extend the functionality of the Hcp1_cys3 gluing unit, Fe3O4 and CoFe2O4 NPs are aligned in a magnetic field and connected by utilization of cysteine-modified Hcp1. After lyophilization, a fiber-like material of micrometer scale length can be observed. The Fe3O4 Hcp1_cys3 fibers show superparamagnetic behavior with a decreasing blocking temperature and an increasing remanent magnetization leading to a higher squareness value of the hysteresis curve. Thus the Hcp1_cys3 unit is shown to be very versatile in the formation of new biohybrid materials with enhanced magnetic, catalytic and optical properties.