Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila.

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

Triblock Proteins with Weakly Dimerizing Terminal Blocks and an Intrinsically Disordered Region for Rational Design of Condensate Properties.

Condensates are molecular assemblies that are formed through liquid-liquid phase separation and play important roles in many biological processes. The rational design of condensate formation and their properties is central to applications, such as biosynthetic materials, synthetic biology, and for understanding cell biology. Protein engineering is used to make a triblock structure with varying terminal blocks of folded proteins on both sides of an intrinsically disordered mid-region. Dissociation constants are determined in the range of micromolar to millimolar for a set of proteins suitable for use as terminal blocks. Varying the weak dimerization of terminal blocks leads to an adjustable tendency for condensate formation while keeping the intrinsically disordered region constant. The dissociation constants of the terminal domains correlate directly with the tendency to undergo liquid-liquid phase separation. Differences in physical properties, such as diffusion rate are not directly correlated with the strength of dimerization but can be understood from the properties and interplay of the constituent blocks. The work demonstrates the importance of weak interactions in condensate formation and shows a principle for protein design that will help in fabricating functional condensates in a predictable and rational way.

Effect of Phosphate on the Molecular Properties, Interactions, and Assembly of Engineered Spider Silk Proteins.

Phosphate plays a vital role in spider silk spinning and has been utilized in numerous artificial silk spinning attempts to replicate the remarkable mechanical properties of natural silk fiber. Its application in artificial processes has, however, yielded varying outcomes. It is thus necessary to investigate the origins and mechanisms behind these differences. By using recombinant silk protein SC-ADF3 derived from the garden spider Araneus diadematus, here, we describe its conformational changes under various conditions, elucidating the effect of phosphate on SC-ADF3 silk protein properties and interactions. Our results demonstrate that elevated phosphate levels induce the irreversible conformational conversion of SC-ADF3 from random coils to ß-sheet structures, leading to decreased protein solubility over time. Furthermore, exposure of SC-ADF3 to phosphate stiffens already formed structures and reduces the ability to form new interactions. Our findings offer insights into the underlying mechanism through which phosphate-induced ß-sheet structures in ADF3-related silk proteins impede fiber formation in the subsequent phases. From a broader perspective, our studies emphasize the significance of silk protein conformation for functional material formation, highlighting that the formation of ß-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.

Exploration of Chemical Diversity in Intercellular Quorum Sensing Signalling Systems in Prokaryotes.

Quorum sensing (QS) serves as a vital means of intercellular signalling in a variety of prokaryotes, which enables single cells to act in multicellular configurations. The potential to control community-wide responses has also sparked numerous recent biotechnological innovations. However, our capacity to utilize intercellular communication is hindered due to a scarcity of complementary signalling systems and a restricted comprehension of interconnections between these systems caused by variations in their dynamic range. In this study, we utilize uniform manifold approximation and projection and extended-connectivity fingerprints to explore the available chemical space of QS signalling molecules. We investigate and experimentally characterize a set of closely related QS signalling ligands, consisting of N-acyl homoserine lactones and the aryl homoserine lactone p-coumaroyl, as well as a set of more widely diverging QS ligands, consisting of photopyrones, dialkylresorcinols, 3,5-dimethylpyrazin-2-ol and autoinducer-2, and define their performance. We report on a set of six signal- and promoter-orthogonal intercellular QS signalling systems, significantly expanding the toolkit for engineering community-wide behaviour. Furthermore, we demonstrate that ligand diversity can serve as a statistically significant tool to predict much more complicated ligand-receptor interactions. This approach highlights the potential of dimensionality reduction to explore chemical diversity in microbial dynamics.

Highly Hydrophobic Films of Engineered Silk Proteins by a Simple Deposition Method.

Molecular engineering of protein structures offers a uniquely versatile route for novel functionalities in materials. Here, we describe a method to form highly hydrophobic thin films using genetically engineered spider silk proteins. We used structurally engineered protein variants containing ADF3 and AQ12 spider silk sequences. Wetting properties were studied using static and dynamic contact angle measurements. Solution conditions and the surrounding humidity during film preparation were key parameters to obtain high hydrophobicity, as shown by contact angles in excess of 120°. Although the surface layer was highly hydrophobic, its structure was disrupted by the added water droplets. Crystal-like structures were found at the spots where water droplets had been placed. To understand the mechanism of film formation, different variants of the proteins, the topography of the films, and secondary structures of the protein components were studied. The high contact angle in the films demonstrates that the conformations that silk proteins take in the protein layer very efficiently expose their hydrophobic segments. This work reveals a highly amphiphilic nature of silk proteins and contributes to an understanding of their assembly mechanisms. It will also help in designing diverse technical uses for recombinant silk.

Shaping Liquid Droplets on an Active Air-Ferrofluid Interface.

An air-liquid interface is important in many biological and industrial applications, where the manipulation of liquids on the air-liquid interface can have a significant impact. However, current manipulation techniques on the interface are mostly limited to transportation and trapping. Here, we report a magnetic liquid shaping method that can squeeze, rotate, and shape nonmagnetic liquids on an air-ferrofluid interface with programmable deformation. We can control the aspect ratio of the ellipse and generate repeatable quasi-static shapes of a hexadecane oil droplet. We can rotate droplets and stir liquids into spiral-like structures. We can also shape phase-changing liquids and fabricate shape-programmed thin films at the air-ferrofluid interface. The proposed method may potentially open up new possibilities for film fabrication, tissue engineering, and biological experiments that can be carried out at an air-liquid interface.

Spidroins under the Influence of Alcohol: Effect of Ethanol on Secondary Structure and Molecular Level Solvation of Silk-Like Proteins.

Future sustainable materials based on designer biomolecules require control of the solution assembly, but also interfacial interactions. Alcohol treatments of protein materials are an accessible means to this, making understanding of the process at the molecular level of seminal importance. We focus here on the influence of ethanol on spidroins, the main proteins of silk. By large-scale atomistically detailed molecular dynamics (MD) simulations and interconnected experiments, we characterize the protein aggregation, secondary structure changes, molecular level origins of them, and solvation environment changes for the proteins, as induced by ethanol as a solvation additive. The MD and circular dichoroism (CD) findings jointly show that ethanol promotes ordered structure in the protein molecules, leading to an increase of helix content and turns but also increased aggregation, as revealed by dynamic light scattering (DLS) and light microscopy. The structural changes correlate at the molecular level with increased intramolecular hydrogen bonding. The simulations reveal that polar amino acids, such as glutamine and serine, are most influenced by ethanol, whereas glycine residues are most prone to be involved in the ethanol-induced secondary structure changes. Furthermore, ethanol engages in interactions with the hydrophobic alanine-rich regions of the spidroin, significantly decreasing the hydrophobic interactions of the protein with itself and its surroundings. The protein solutes also change the microstructure of water/ethanol mixtures, essentially decreasing the level of larger local clustering. Overall, the work presents a systematic characterization of ethanol effects on a widely used, common protein type, spidroins, and generalizes the findings to other intrinsically disordered proteins by pinpointing the general features of the response. The results can aid in designing effective alcohol treatments for proteins, but also enable design and tuning of protein material properties by a relatively controllable solvation handle, the addition of ethanol.

pH dependence of the assembly mechanism and properties of poly(L-lysine) and poly(L-glutamic acid) complexes.

We show by extensive experimental characterization combined with molecular simulations that pH has a major impact on the assembly mechanism and properties of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) complexes. A combination of dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) is used to assess the complexation, charge state, and other physical characteristics of the complexes, isothermal titration calorimetry (ITC) is used to examine the complexation thermodynamics, and circular dichroism (CD) is used to extract the polypeptides' secondary structure. For enhanced analysis and interpretation of the data, analytical ultracentrifugation (AUC) is used to define the precise molecular weights and solution association of the peptides. Molecular dynamics simulations reveal the associated intra- and intermolecular binding changes in terms of intrinsic vs. extrinsic charge compensation, the role of hydrogen bonding, and secondary structure changes, aiding in the interpretation of the experimental data. We combine the data to reveal the pH dependency of PLL/PGA complexation and the associated molecular level mechanisms. This work shows that not only pH provides a means to control complex formation but also that the associated changes in the secondary structure and binding conformation can be systematically used to control materials assembly. This gives access to rational design of peptide materials via pH control.

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins.

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods.

Emergence of Elastic Properties in a Minimalist Resilin-Derived Heptapeptide upon Bromination.

Bromination is herein exploited to promote the emergence of elastic behavior in a short peptide-SDSYGAP-derived from resilin, a rubber-like protein exerting its role in the jumping and flight systems of insects. Elastic and resilient hydrogels are obtained, which also show self-healing behavior, thanks to the promoted non-covalent interactions that limit deformations and contribute to the structural recovery of the peptide-based hydrogel. In particular, halogen bonds may stabilize the ß-sheet organization working as non-covalent cross-links between nearby peptide strands. Importantly, the unmodified peptide (i.e., wild type) does not show such properties. Thus, SDSY(3,5-Br)GAP is a novel minimalist peptide elastomer.

Liquid-Liquid Phase Separation and Assembly of Silk-like Proteins is Dependent on the Polymer Length.

Phase transitions have an essential role in the assembly of nature's protein-based materials into hierarchically organized structures, yet many of the underlying mechanisms and interactions remain to be resolved. A central question for designing proteins for materials is how the protein architecture and sequence affects the nature of the phase transitions and resulting assembly. In this work, we produced 82 kDa (1×), 143 kDa (2×), and 204 kDa (3×) silk-mimicking proteins by taking advantage of protein ligation by SpyCatcher/Tag protein-peptide pair. We show that the three silk proteins all undergo a phase transition from homogeneous solution to assembly formation. In the assembly phase, a length- and concentration-dependent transition between two distinct assembly morphologies, one forming aggregates and another coacervates, exists. The coacervates showed properties that were dependent on the protein size. Computational modeling of the proteins by a bead-spring model supports the experimental results and provides us a possible mechanistic origin for the assembly transitions based on architectures and interactions.

Self-Assembly of Silk-like Protein into Nanoscale Bicontinuous Networks under Phase-Separation Conditions.

Liquid-liquid phase separation of biomacromolecules is crucial in various inter- and extracellular biological functions. This includes formation of condensates to control, e.g., biochemical reactions and structural assembly. The same phenomenon is also found to be critically important in protein-based high-performance biological materials. Here, we use a well-characterized model triblock protein system to demonstrate the molecular level formation mechanism and structure of its condensate. Large-scale molecular modeling supported by analytical ultracentrifuge characterization combined with our earlier high magnification precision cryo-SEM microscopy imaging leads to deducing that the condensate has a bicontinuous network structure. The bicontinuous network rises from the proteins having a combination of sites with stronger mutual attraction and multiple weakly attractive regions connected by flexible, multiconfigurational linker regions. These attractive sites and regions behave as stickers of varying adhesion strength. For the examined model triblock protein construct, the ß-sheet-rich end units are the stronger stickers, while additional weaker stickers, contributing to the condensation affinity, rise from spring-like connections in the flexible middle region of the protein. The combination of stronger and weaker sticker-like connections and the flexible regions between the stickers result in a versatile, liquid-like, self-healing structure. This structure also explains the high flexibility, easy deformability, and diffusion of the proteins, decreasing only 10-100 times in the bicontinuous network formed in the condensate phase in comparison to dilute protein solution. The here demonstrated structure and condensation mechanism of a model triblock protein construct via a combination of the stronger binding regions and the weaker, flexible sacrificial-bond-like network as well as its generalizability via polymer sticker models provide means to not only understand intracellular organization, regulation, and cellular function but also to identify direct control factors for and to enable engineering improved protein and polymer constructs to enhance control of advanced fiber materials, smart liquid biointerfaces, or self-healing matrices for pharmaceutics or bioengineering materials.

Modulating the Mechanical Performance of Macroscale Fibers through Shear-Induced Alignment and Assembly of Protein Nanofibrils.

Protein-based fibers are used by nature as high-performance materials in a wide range of applications, including providing structural support, creating thermal insulation, and generating underwater adhesives. Such fibers are commonly generated through a hierarchical self-assembly process, where the molecular building blocks are geometrically confined and aligned along the fiber axis to provide a high level of structural robustness. Here, this approach is mimicked by using a microfluidic spinning method to enable precise control over multiscale order during the assembly process of nanoscale protein nanofibrils into micro- and macroscale fibers. By varying the flow rates on chip, the degree of nanofibril alignment can be tuned, leading to an orientation index comparable to that of native silk. It is found that the Young's modulus of the resulting fibers increases with an increasing level of nanoscale alignment of the building blocks, suggesting that the mechanical properties of macroscopic fibers can be controlled through varying the level of ordering of the nanoscale building blocks. Capitalizing on strategies evolved by nature, the fabrication method allows for the controlled formation of macroscopic fibers and offers the potential to be applied for the generation of further novel bioinspired materials.

Three-Dimensional Printed Cell Culture Model Based on Spherical Colloidal Lignin Particles and Cellulose Nanofibril-Alginate Hydrogel.

Three-dimensional (3D) printing has been an emerging technique to fabricate precise scaffolds for biomedical applications. Cellulose nanofibril (CNF) hydrogels have attracted considerable attention as a material for 3D printing because of their shear-thinning properties. Combining cellulose nanofibril hydrogels with alginate is an effective method to enable cross-linking of the printed scaffolds in the presence of Ca2+ ions. In this work, spherical colloidal lignin particles (CLPs, also known as spherical lignin nanoparticles) were used to prepare CNF-alginate-CLP nanocomposite scaffolds. High-resolution images obtained by atomic force microscopy (AFM) showed that CLPs were homogeneously mixed with the CNF hydrogel. CLPs brought antioxidant properties to the CNF-alginate-CLP scaffolds in a concentration-dependent manner and increased the viscosity of the hydrogels at a low shear rate, which correspondingly provide better shape fidelity and printing resolution to the scaffolds. Interestingly, the CLPs did not affect the viscosity at high shear rates, showing that the shear thinning behavior typical for CNF hydrogels was retained, enabling easy printing. The CNF-alginate-CLP scaffolds demonstrated shape stability after printing, cross-linking, and storage in Dulbecco's phosphate buffer solution (DPBS +) containing Ca2+ and Mg2+ ions, up to 7 days. The 3D-printed scaffolds showed relative rehydration ratio values above 80% after freeze-drying, demonstrating a high water-retaining capability. Cell viability tests using hepatocellular carcinoma cell line HepG2 showed no negative effect of CLPs on cell proliferation. Fluorescence microscopy indicated that HepG2 cells grew not only on the surfaces but also inside the porous scaffolds. Overall, our results demonstrate that nanocomposite CNF-alginate-CLP scaffolds have high potential in soft-tissue engineering and regenerative-medicine applications.

Dynamic Assembly of Class II Hydrophobins from T. reesei at the Air-Water Interface.

Class II hydrophobins are amphiphilic proteins produced by filamentous fungi. One of their typical features is the tendency to accumulate at the interface between an aqueous phase and a hydrophobic phase, such as the air-water interface. The kinetics of the interfacial self-assembly of wild-type hydrophobins HFBI and HFBII and some of their engineered variants at the air-water interface were measured by monitoring the accumulated mass at the interface via nondestructive ellipsometry measurements. The resulting mass vs time curves revealed unusual kinetics for a monolayer formation that did not follow a typical Langmuir-type of behavior but had a rather coverage-independent rate instead. Typically, the full surface coverage was obtained at masses corresponding to a monolayer. The formation of multilayers was not observed. Atomic force microscopy revealed formation and growth of non-fusing protein clusters at the interface. The mechanism of the adsorption was studied by varying the structure or charges of the protein or the ionic strength of the subphase, revealing that the lateral interactions between the hydrophobins play a role in their interfacial assembly. Additionally, a theoretical model was introduced to identify the underlying mechanism of the unconventional adsorption kinetics.

Binding Forces of Cellulose Binding Modules on Cellulosic Nanomaterials.

In this study, the interaction forces between different cellulosic nanomaterials and a protein domain belonging to cellulose binding modules family 1 (CBM1) were investigated at the molecular scale. Cellulose binding modules are protein domains found in carbohydrate active enzymes having an affinity toward cellulosic materials. Here, the binding force of a fusion protein containing a cellulose binding module (CBM1) produced recombinantly in E. coli was quantified on different cellulose nanocrystals immobilized on surfaces. Adhesion of the CBM on cellulose with different degrees of crystallinity as well as on chitin nanocrystals was examined. This study was carried out by single molecule force spectroscopy using an atomic force microscope, which enables the detection of binding force of individual molecules. The study contains a preliminary quantification of the interactions at the molecular level that sheds light on the development of new nanocellulose-based nanocomposites with improved strength and elasticity.

EGFR in Tumor-Associated Myeloid Cells Promotes Development of Colorectal Cancer in Mice and Associates With Outcomes of Patients.

BACKGROUND & AIMS: Inhibitors of the epidermal growth factor receptor (EGFR) are the first-line therapy for patients with metastatic colorectal tumors without RAS mutations. However, EGFR inhibitors are ineffective in these patients, and tumor level of EGFR does not associate with response to therapy. We screened human colorectal tumors for EGFR-positive myeloid cells and investigated their association with patient outcome. We also performed studies in mice to evaluate how EGFR expression in tumor cells and myeloid cells contributes to development of colitis-associated cancer and ApcMin-dependent intestinal tumorigenesis. METHODS: We performed immunohistochemical and immunofluorescent analyses of 116 colorectal tumor biopsies to determine levels of EGFR in tumor and stroma; we also collected information on tumor stage and patient features and outcomes. We used the Mann-Whitney U and Kruskal-Wallis tests to correlate tumor levels of EGFR with tumor stage, and the Kaplan-Meier method to estimate patients' median survival time. We performed experiments in mice lacking EGFR in intestinal epithelial cells (Villin-Cre; Egfrf/f and Villin-CreERT2; Egfrf/f mice) or myeloid cells (LysM-Cre; Egfrf/f mice) on a mixed background. These mice were bred with ApcMin/+ mice; colitis-associated cancer and colitis were induced by administration of dextran sodium sulfate (DSS), with or without azoxymethane (AOM), respectively. Villin-CreERT2 was activated in developed tumors by administration of tamoxifen to mice. Littermates that expressed full-length EGFR were used as controls. Intestinal tissues were collected; severity of colitis, numbers and size of tumors, and intestinal barrier integrity were assessed by histologic, immunohistochemical, quantitative reverse transcription polymerase chain reaction, and flow cytometry analyses. RESULTS: We detected EGFR in myeloid cells in the stroma of human colorectal tumors; myeloid cell expression of EGFR associated with tumor metastasis and shorter patient survival time. Mice with deletion of EGFR from myeloid cells formed significantly fewer and smaller tumors than the respective EGFR-expressing controls in an ApcMin/+ background as well as after administration of AOM and DSS. Deletion of EGFR from intestinal epithelial cells did not affect tumor growth. Furthermore, tamoxifen-induced deletion of EGFR from epithelial cells of established intestinal tumors in mice given AOM and DSS did not reduce tumor size. EGFR signaling in myeloid cells promoted activation of STAT3 and expression of survivin in intestinal tumor cells. Mice with deletion of EGFR from myeloid cells developed more severe colitis after DSS administration, characterized by increased intestinal inflammation and intestinal barrier disruption, than control mice or mice with deletion of EGFR from intestinal epithelial cells. EGFR-deficient myeloid cells in the colon of DSS-treated LysM-Cre; Egfrf/f mice had reduced expression of interleukin 6 (IL6), and epithelial STAT3 activation was reduced compared with controls. Administration of recombinant IL6 to LysM-Cre; Egfrf/f mice given DSS protected them from weight loss and restored epithelial proliferation and STAT3 activation, compared with administration of DSS alone to these mice. CONCLUSIONS: Increased expression of EGFR in myeloid cells from the colorectal tumor stroma associates with tumor progression and reduced survival time of patients with metastatic colorectal cancer. Deletion of EGFR from myeloid cells, but not intestinal epithelial cells, protects mice from colitis-induced intestinal cancer and ApcMin-dependent intestinal tumorigenesis. Myeloid cell expression of EGFR increases activation of STAT3 and expression of survivin in intestinal epithelial cells and expression of IL6 in colon tissues. These findings indicate that expression of EGFR by myeloid cells of the colorectal tumor stroma, rather than the cancer cells themselves, contributes to tumor development.

In-solution antibody harvesting with a plant-produced hydrophobin-Protein A fusion.

Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35-fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB-induced protein body formation. We also demonstrated production of HFBI-Protein A fusion protein in tobacco BY-2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody-binding capacity of the Protein A block was similar to the nonfused Protein A. The best-performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two-phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.

Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism.

The mechanism of silk assembly, and thus the cues for the extraordinary properties of silk, can be explored by studying the simplest protein parts needed for the formation of silk-like materials. The recombinant spider silk protein 4RepCT, consisting of four repeats of polyalanine and glycine-rich segments (4Rep) and a globular C-terminal domain (CT), has previously been shown to assemble into silk-like fibers at the liquid-air interface. Herein, we study the interfacial behavior of the two parts of 4RepCT, revealing new details on how each protein part is crucial for the silk assembly. Interfacial rheology and quartz crystal microbalance with dissipation show that 4Rep interacts readily at the interfaces. However, organized nanofibrillar structures are formed only when 4Rep is fused to CT. A strong interplay between the parts to direct the assembly is demonstrated. The presence of either a liquid-air or a liquid-solid interface had a surprisingly similar influence on the assembly.