Self‐Assembling Proteins as High‐Performance Substrates for Embryonic Stem Cell Self‐Renewal

The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self‐renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large‐scale cell production under xeno‐free culture conditions using current matrices. Here, a bioactive, recombinant, protein‐based polymer, termed ZT^Fn, is presented that closely mimics human plasma fibronectin and serves as an economical, xeno‐free, biodegradable, and functionally adaptable cell substrate. The ZT^Fn substrate supports with high performance the propagation and long‐term self‐renewal of human embryonic stem cells while preserving their pluripotency. The ZT^Fn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.     

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.     

Ligand‐Free Fe3O4/CMCS Nanoclusters with Negative Charges for Efficient Structure‐Selective Protein Adsorption

The easy and effective capture of a single protein from a complex mixture is of great significance in proteomics and diagnostics. However, adsorbing nanomaterials are commonly decorated with specific ligands through a complicated and arduous process. Fe₃O₄/carboxymethylated chitosan (Fe₃O₄/CMCS) nanoclusters are developed as a new nonligand modified strategy to selectively capture bovine hemoglogin (BHB) and other structurally similar proteins (i.e., lysozyme (LYZ) and chymotrypsin (CTP)). The ligand‐free Fe₃O₄/CMCS nanoclusters, in addition to their simple and economical two‐step preparation process, possess many merits, including uniform morphology, high negative charges (?27 mV), high saturation magnetization (60 emu g^?1), and high magnetic content (85%). Additionally, the ligand‐free Fe₃O₄/CMCS nanoclusters are found to selectively capture BHB in a model protein mixture even within biological samples. The reason for selective protein capture is further investigated from nanomaterials and protein structure. In terms of nanomaterials, it is found that high negative charges are conducive to selectively adsorb BHB. In consideration of protein structure, interestingly, the ligand‐free magnetic nanoclusters display a structure‐selective protein adsorption capacity to efficiently capture other proteins structurally similar to BHB, such as LYZ and CTP, showing great potential of the ligand‐free strategy in biomedical field.     

Biodegradable Protein‐based Films and Their Properties: A Comparative Study

Biodegradable protein‐based films prepared from different protein sources [commercial bovine gelatine (CG), giant catfish skin gelatine (GG), soy protein isolates (SPI), fish myofibrillar protein (FMP) and whey protein concentrate (WPC)] were all investigated for their mechanical, physical, chemical, thermal and barrier properties. The properties of the resulting films were then compared with those of commercial wrap films [polyvinyl chloride (PVC)]. The film forming solution containing 7% (w/v) protein and 50% (w/w) glycerol was used to produce the films through a casting method. Of the protein‐based films, the GG film had the highest tensile strength and elongation, while the WPC film exhibited the lowest film solubility, water vapour permeability, light transmission in UV‐Vis range (200–800 nm) and film transparency. However, the colour of the FMP film and the thickness were closer to that of the PVC film, particularly the L* and b* values. The appearances of the protein‐based films were similar to the PVC film, and they were uniformly transparent. Therefore, biodegradable films produced from different types of protein sources exhibited differences in their properties. These results are consistent with data from FTIR and protein pattern analyses. Based on these findings, different sources of protein‐based films can be used as an alternative for food packaging applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Engineering Protein Nanoparticles Out from Components of the Human Microbiome

Nanoscale protein materials are highly convenient as vehicles for targeted drug delivery because of their structural and functional versatility. Selective binding to specific cell surface receptors and penetration into target cells require the use of targeting peptides. Such homing stretches should be incorporated to larger proteins that do not interact with body components, to prevent undesired drug release into nontarget organs. Because of their low interactivity with human body components and their tolerated immunogenicity, proteins derived from the human microbiome are appealing and fully biocompatible building blocks for the biofabrication of nonreactive, inert protein materials within the nanoscale. Several phage and phage‐like bacterial proteins with natural structural roles are produced in Escherichia coli as polyhistidine‐tagged recombinant proteins, looking for their organization as discrete, nanoscale particulate materials. While all of them self‐assemble in a variety of sizes, the stability of the resulting constructs at 37 °C is found to be severely compromised. However, the fine adjustment of temperature and Zn²⁺ concentration allows the formation of robust nanomaterials, fully stable in complex media and under physiological conditions. Then, microbiome‐derived proteins show promise for the regulatable construction of scaffold protein nanomaterials, which can be tailored and strengthened by simple physicochemical approaches.     

A Supramolecular Approach to Enzyme Immobilization in Micro‐Channels

A supramolecular assembly scheme is developed to enable the facile in‐situ immobilization of enzymes in a microfluidic channel system. A combination of orthogonal supramolecular interactions of host (β‐cyclodextrin)–guest (adamantane) and biotin–Streptavidin (SAv) interactions are employed to generate reusable homogeneous enzyme layers in microchannels. The structural integrity and catalytic activity of the immobilized enzyme calf‐intestine alkaline phosphatase (AlkPh) is demonstrated. From the kinetic analysis of a dephosphorylation reaction, the specificity constant k_cat/K_M for immobilized alkaline phosphatase in the channels is on the order of 10⁵ M^?1s^?1 and comparable to known literature values in other environments. These observations are ascribed to the good access of the substrate to favorably oriented enzymes across the microchannel. Therefore, this study demonstrates the great potential for adopting a supramolecular assembly scheme to immobilize enzymes in microfluidic devices.     

One‐Pot Synthesis of Multiple Protein‐Encapsulated DNA Flowers and Their Application in Intracellular Protein Delivery

Inspired by biological systems, many biomimetic methods suggest fabrication of functional materials with unique physicochemical properties. Such methods frequently generate organic–inorganic composites that feature highly ordered hierarchical structures with intriguing properties, distinct from their individual components. A striking example is that of DNA–inorganic hybrid micro/nanostructures, fabricated by the rolling circle technique. Here, a novel concept for the encapsulation of bioactive proteins in DNA flowers (DNF) while maintaining the activity of protein payloads is reported. A wide range of proteins, including enzymes, can be simultaneously associated with the growing DNA strands and Mg₂PPi crystals during the rolling circle process, ultimately leading to the direct immobilization of proteins into DNF. The unique porous structure of this construct, along with the abundance of Mg ions and DNA molecules present, provides many interaction sites for proteins, enabling high loading efficiency and enhanced stability. Further, as a proof of concept, it is demonstrated that the DNF can deliver payloads of cytotoxic protein (i.e., RNase A) to the cells without a loss in its biological function and structural integrity, resulting in highly increased cell death compared to the free protein.     

Surface Dependence of Protein Nanocrystal Formation

The self‐assembly kinetics and nanocrystal formation of the bacterial surface‐layer‐protein SbpA are studied with a combination of quartz crystal microbalance with dissipation monitoring (QCM‐D) and atomic force microscopy (AFM). Silane coupling agents, aminopropyltriethoxysilane (APTS) and octadecyltrichlorosilane (OTS), are used to vary the protein–surface interaction in order to induce new recrystallization pathways. The results show that the final S‐layer crystal lattice parameters (a = b = 14 nm, γ = 90°), the layer thickness (15 nm), and the adsorbed mass density (1700 ng cm^?2) are independent of the surface chemistry. Nevertheless, the adsorption rate is five times faster on APTS and OTS than on SiO_2, strongly affecting protein nucleation and growth. As a consequence, protein crystalline domains of 0.02 µm² for APTS and 0.05 µm² for OTS are formed, while for silicon dioxide the protein domains have a typical size of about 32 µm². In addition, more‐rigid crystalline protein layers are formed on hydrophobic substrates. In situ AFM experiments reveal three different kinetic steps: adsorption, self‐assembly, and crystalline‐domain reorganization. These steps are corroborated by frequency–dissipation curves. Finally, it is shown that protein adsorption is a diffusion‐driven process. Experiments at different protein concentrations demonstrate that protein adsorption saturates at 0.05 mg mL^?1 on silane‐coated substrates and at 0.07 mg mL^?1 on hydrophilic silicon dioxide.     

Comparison of serum albumin,C‐reactive protein and carotid atherosclerosis as predictors of 10‐year mortality in hemodialysis patients

Serum albumin, C‐reactive protein (CRP), and the intima‐medial thickness of the common carotid artery (CA‐IMT) are associated with clinical outcomes in hemodialysis (HD) patients. However, it remains unclear which parameters are more reliable as predictors of long‐term mortality. We measured serum albumin, CRP, and CA‐IMT in 206 HD patients younger than 80 years old, and followed them for the next 10 years. One hundred sixty‐eight patients (age: 57 ± 11 years, time on HD: 11 ± 7 years) were enrolled in the analyses. We divided all patients into three tertiles according to their albumin levels, and conducted multivariate analyses to examine the impact on 10‐year mortality. Seventy‐three (43.5%) patients had expired during the follow‐up. Serum albumin was significantly lower in the expired patients than in the surviving patients (3.8 ± 0.3 vs. 4.0 ± 0.3, P<0.01), while CRP (4.7 ± 5.0 vs. 2.8 ± 3.5 g/L, P=0.01) and CA‐IMT (0.70 ± 0.15 vs. 0.59 ± 0.11 mm, P<0.01) were significantly higher in the expired group. The multivariate analysis revealed that there was a significantly higher risk for total mortality in HD patients with serum albumin <3.8 g/dL (odds ratio 5.04 [95% CI: 1.30–19.60], P=0.02) when compared with those with albumin >4.1 g/dL. In contrast, CRP and CA‐IMT did not associate with total death. It follows from these findings that serum albumin is more superior as a mortality predictor compared with CRP and CA‐IMT in HD patients.     

Recombinant glycans on an S-layer self-assembly protein: a new dimension for nanopatterned biomaterials

Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S-layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self-assembly protein as a cellular prerequisite for protein glycosylation, and on engineering of one of the natural protein O-glycosylation sites into a target for N-glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co- or post-translationally transferred to the S-layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the S-layer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S-layer protein self-assembly system. Tailor-made ("functional") nanopatterned, self-assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics.     

Higher Order Assembly of Virus‐like Particles (VLPs) Mediated by Multi‐valent Protein Linkers

Two‐ and three‐dimensional assembly of nanoparticles has generated significant interest because these higher order structures could exhibit collective behaviors/properties beyond those of the individual nanoparticles. Highly specific interactions between molecules, which biology exploits to regulate molecular assemblies such as DNA hybridization, often provide inspiration for the construction of higher order materials using bottom‐up approaches. In this study, higher order assembly of virus‐like particles (VLPs) derived from the bacteriophage P22 is demonstrated by using a small adaptor protein, Dec, which binds to symmetry specific sites on the P22 capsid. Two types of connector proteins, which have different number of P22 binding sites and different geometries (ditopic linker with liner geometry and tetratopic linker with tetrahedral geometry) have been engineered through either a point mutation of Dec or genetic fusion with another protein, respectively. Bulk assembly and layer‐by‐layer deposition of P22 VLPs from solution was successfully achieved using both of the engineered multi‐topic linker molecules, while Dec with only a single binding site does not mediate P22 assembly. Beyond the two types of linkers developed in this study, a wide range of different connector geometries could be envisioned using a similar engineering approach. This is a powerful strategy to construct higher order assemblies of VLP based nanomaterials.