Medium chain length polyhydroxyalkanoate produced from ethanol by Pseudomonas putida grown in liquid obtained from acidogenic digestion of organic municipal solid waste.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) up to about 6 g.L-1 was obtained by feeding ethanol to Pseudomonas putida growing in liquid obtained from acidogenic digestion of organic municipal solid waste. Washing the wet, heat-inactivated Pseudomonas cells at the end of the fermentation with ethanol obviated the need of drying the biomass and enabled the removal of contaminating lipids before solvent-mediated extraction of PHA. Using 'green' solvents, 90 to near 100% of the mcl-PHA was extracted and purities of 71-78% mcl-PHA were reached already by centrifugation and decantation without further filtration for biomass removal. The mcl-PHA produced in this way consists of 10-18% C8, 72-78% C10 and 8-12% C12 chains (entirely medium chain length), has a crystallinity and melting temperature of ∼13% and ∼49 °C, respectively, and is a stiff rubberlike, colourless material at room temperature.

Production of protein-based polymers in Pichia pastoris.

Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris. We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e., the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo. Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.

Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain.

Emerging DNA-based nanotechnologies would benefit from the ability to modulate the properties (e.g., solubility, melting temperature, chemical stability) of diverse DNA templates (single molecules or origami nanostructures) through controlled, self-assembling coatings. We here introduce a DNA coating agent, called C8-BSso7d, which binds to and coats with high specificity and affinity, individual DNA molecules as well as folded origami nanostructures. C8-BSso7d coats and protects without condensing, collapsing or destroying the spatial structure of the underlying DNA template. C8-BSso7d combines the specific nonelectrostatic DNA binding affinity of an archeal-derived DNA binding domain (Sso7d, 7 kDa) with a long hydrophilic random coil polypeptide (C8, 73 kDa), which provides colloidal stability (solubility) through formation of polymer brushes around the DNA templates. C8-BSso7d is produced recombinantly in yeast and has a precise (but engineerable) amino acid sequence of precise length. Using electrophoresis, AFM, and fluorescence microscopy we demonstrate protein coat formation with stiffening of one-dimensional templates (linear dsDNA, supercoiled dsDNA and circular ssDNA), as well as coat formation without any structural distortion or disruption of two-dimensional DNA origami template. Combining the programmability of DNA with the nonperturbing precise coating capability of the engineered protein C8-BSso7d holds promise for future applications such as the creation of DNA-protein hybrid networks, or the efficient transfection of individual DNA nanostructures into cells.

Cross-Linking and Bundling of Self-Assembled Protein-Based Polymer Fibrils via Heterodimeric Coiled Coils.

Previously, we developed triblock protein polymers that form fibrillar hydrogels at low protein polymer concentrations (denoted C2-SH48-C2). We here demonstrate that the structure of these hydrogels can be tuned via heterodimeric coiled coils that cross-link and bundle the self-assembled protein polymer fibrils. We fused well-characterized, 47 amino acids-long heterodimeric coiled coil "linkers" (DA or DB) to the C-terminus of the triblock polymer. The resulting C2-SH48-C2-DA and C2-SH48-C2-DB polymers, were successfully produced as secreted proteins in Pichia pastoris, with titers of purified protein in the order of g L-1 of clarified broth. Atomic force microscopy showed that fibrils formed by either C2-SH48-C2-DA or C2-SH48-C2-DB alone already displayed extensive bundling, apparently as a result of homotypic (DA/DA and DB/DB) interactions. For fibrils prepared from protein polymers having no linkers, plus a small fraction of polymers containing either DA or DB linkers, no cross-linking and bundling was observed. At these same low concentrations of linkers, fibrils containing both the DA and the DB linkers did show cross-linking and bundling as a consequence of heterodimer formation. This work shows that we can control the extent of bundling and cross-linking of supramolecular fibrils by varying the density of heterodimerizing coiled coils in the fibrils, which is promising for the further development of materials that mimic the extracellular matrix.

Nanofibrillar hydrogel scaffolds from recombinant protein-based polymers with integrin- and proteoglycan-binding domains.

This study describes the design, production, and testing of functionalized variants of a recombinant protein-based polymer that forms nanofibrillar hydrogels with self-healing properties. With a view to bone tissue engineering applications, we equipped these variants with N-terminal extensions containing either (1) integrin-binding (RGD) or (2) less commonly studied proteoglycan-binding (KRSR) cell-adhesive motifs. The polymers were efficiently produced as secreted proteins using the yeast Pichia pastoris and were essentially monodisperse. The pH-responsive protein-based polymers are soluble at low pH and self-assemble into supramolecular fibrils and hydrogels at physiological pH. By mixing functionalized and nonfunctionalized proteins in different ratios, and adjusting pH, hydrogel scaffolds with the same protein concentration but varying content of the two types of cell-adhesive motifs were readily obtained. The scaffolds were used for the two-dimensional culture of MG-63 osteoblastic cells. RGD domains had a slightly stronger effect than KRSR domains on adhesion, activity, and spreading. However, scaffolds featuring both functional domains revealed a clear synergistic effect on cell metabolic activity and spreading, and provided the highest final degree of cell confluency. The mixed functionalized hydrogels presented here thus allowed to tailor the osteoblastic cell response, offering prospects for their further development as scaffolds for bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3082-3092, 2016.

Enhanced stiffness of silk-like fibers by loop formation in the corona leads to stronger gels.

We study the self-assembly of protein polymers consisting of a silk-like block flanked by two hydrophilic blocks, with a cysteine residue attached to the C-terminal end. The silk blocks self-assemble to form fibers while the hydrophilic blocks form a stabilizing corona. Entanglement of the fibers leads to the formation of hydrogels. Under oxidizing conditions the cysteine residues form disulfide bridges, effectively connecting two corona chains at their ends to form a loop. We find that this leads to a significant increase in the elastic modulus of the gels. Using atomic force microscopy, we show that this stiffening is due to an increase of the persistence length of the fibers. Self-consistent-field calculations indicate a slight decrease of the lateral pressure in the corona upon loop formation. We argue that this small decrease in the repulsive interactions affects the stacking of the silk-like blocks in the core, resulting in a more rigid fiber.

Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains.

BACKGROUND: Specific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting peptides into the genetic design of the protein polymers. An example of such interaction is the binding of particular proline-rich ligands by so-called WW-domains. In this study, we investigated whether these domains can be produced in the yeast Pichia pastoris as part of otherwise non-interacting protein polymers, and whether they bring about polymer coupling upon mixing. RESULTS: We constructed two variants of a highly hydrophilic protein-based polymer that differ only in their C-terminal extensions. One carries a C-terminal WW domain, and the other a C-terminal proline-rich ligand (PPxY). Both polymers were produced in P. pastoris with a purified protein yield of more than 2 g L(-1) of cell-free broth. The proline-rich module was found to be O-glycosylated, and uncommonly a large portion of the attached oligosaccharides was phosphorylated. Glycosylation was overcome by introducing a Ser → Ala mutation in the PPxY peptide. Tryptophan fluorescence monitored during titration of the polymer containing the WW domain with either the glycosylated or nonglycosylated PPxY-containing polymer revealed binding. The complementary polymers associated with a Kd of ~3 µM, regardless of glycosylation state of the PPxY domain. Binding was confirmed by isothermal titration calorimetry, with a Kd of ~9 µM. CONCLUSIONS: This article presents a blueprint for the production in P. pastoris of protein polymers that can be coupled using the noncovalent interaction between WW domains and proline-rich ligands. The availability of this highly specific coupling tool will hereafter allow us to construct various supramolecular structures and biomaterials.

Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach.

Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.

Heparin as a Bundler in a Self-Assembled Fibrous Network of Functionalized Protein-Based Polymers.

Nature shows excellent control over the mechanics of fibrous hydrogels by assembling protein fibers into bundles of well-defined dimensions. Yet, obtaining artificial materials displaying controlled bundling remains a challenge. Here, we developed genetically engineered protein-based polymers functionalized with heparin-binding KRSR domains and show controlled bundling using heparin as a binder. The protein polymer forms fibers upon increasing the pH to physiological values and at higher concentrations fibrous gels. We show that addition of heparin to the protein polymer with incorporated KRSR domains, induces bundling, which results in faster gel formation and stiffer gels. The interactions are expected to be primarily electrostatic and fiber bundling has an optimum when the positive charges of KRSR are approximately in balance with the negative charges of the heparin. Our study suggests that, generally, a straightforward method to control the properties of fibrous gels is to prepare a fiber former with specific binding domains and then simply adding an appropriate amount of binder.

Production in Pichia pastoris of protein-based polymers with small heterodimer-forming blocks.

Some combinations of leucine zipper peptides are capable of forming α-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications.

Reversible Temperature-Switching of Hydrogel Stiffness of Coassembled, Silk-Collagen-Like Hydrogels.

Recombinant protein polymers, which can combine different bioinspired self-assembly motifs in a well-defined block sequence, have large potential as building blocks for making complex, hierarchically structured materials. In this paper we demonstrate the stepwise formation of thermosensitive hydrogels by combination of two distinct, orthogonal self-assembly mechanisms. In the first step, fibers are coassembled from two recombinant protein polymers: (a) a symmetric silk-like block copolymer consisting of a central silk-like block flanked by two soluble random coil blocks and (b) an asymmetric silk-collagen-like block copolymer consisting of a central random-coil block flanked on one side by a silk-like block and on the other side a collagen-like block. In the second step, induced by cooling, the collagen-like blocks form triple helices and thereby cross-link the fibers, leading to hydrogels with a thermo-reversibly switchable stiffness. Our work demonstrates how complex self-assembled materials can be formed through careful control of the self-assembly pathway.

From micelles to fibers: balancing self-assembling and random coiling domains in pH-responsive silk-collagen-like protein-based polymers.

We study the self-assembly of genetically engineered protein-based triblock copolymers consisting of a central pH-responsive silk-like middle block (S(H)n, where S(H) is a silk-like octapeptide, (GA)3GH and n is the number of repeats) flanked by hydrophilic random coil outer blocks (C2). Our previous work has already shown that triblocks with very long midblocks (n = 48) self-assemble into long, stiff protein filaments at pH values where the middle blocks are uncharged. Here we investigate the self-assembly behavior of the triblock copolymers for a range of midblock lengths, n = 8, 16, 24, 48. Upon charge neutralization of S(H)n by adjusting the pH, we find that C2S(H)8C2 and C2S(H)16C2 form spherical micelles, whereas both C2S(H)24C2 and C2S(H)48C2 form protein filaments with a characteristic beta-roll secondary structure of the silk midblocks. Hydrogels formed by C2S(H)48C2 are much stronger and form much faster than those formed by C2S(H)24C2. Enzymatic digestion of much of the hydrophilic outer blocks is used to show that with much of the hydrophilic outer blocks removed, all silk-midblocks are capable of self-assembling into stiff protein filaments. In that case, reduction of the steric repulsion by the hydrophilic outer blocks also leads to extensive fiber bundling. Our results highlight the opposing roles of the hydrophilic outer blocks and central silk-like midblocks in driving protein filament formation. They provide crucial information for future designs of triblock protein-based polymers that form stiff filaments with controlled bundling, that could mimick properties of collagen in the extracellular matrix.

Design and self-assembly of simple coat proteins for artificial viruses.

Viruses are among the simplest biological systems and are highly effective vehicles for the delivery of genetic material into susceptible host cells. Artificial viruses can be used as model systems for providing insights into natural viruses and can be considered a testing ground for developing artificial life. Moreover, they are used in biomedical and biotechnological applications, such as targeted delivery of nucleic acids for gene therapy and as scaffolds in material science. In a natural setting, survival of viruses requires that a significant fraction of the replicated genomes be completely protected by coat proteins. Complete protection of the genome is ensured by a highly cooperative supramolecular process between the coat proteins and the nucleic acids, which is based on reversible, weak and allosteric interactions only. However, incorporating this type of supramolecular cooperativity into artificial viruses remains challenging. Here, we report a rational design for a self-assembling minimal viral coat protein based on simple polypeptide domains. Our coat protein features precise control over the cooperativity of its self-assembly with single DNA molecules to finally form rod-shaped virus-like particles. We confirm the validity of our design principles by showing that the kinetics of self-assembly of our virus-like particles follows a previous model developed for tobacco mosaic virus. We show that our virus-like particles protect DNA against enzymatic degradation and transfect cells with considerable efficiency, making them promising delivery vehicles.

Genetically engineered silk-collagen-like copolymer for biomedical applications: production, characterization and evaluation of cellular response.

Genetically engineered protein polymers (GEPP) are a class of multifunctional materials with precisely controlled molecular structure and property profile. Representing a promising alternative for currently used materials in biomedical applications, GEPP offer multiple benefits over natural and chemically synthesized polymers. However, producing them in sufficient quantities for preclinical research remains challenging. Here, we present results from an in vitro cellular response study of a recombinant protein polymer that is soluble at low pH but self-organizes into supramolecular fibers and physical hydrogels at neutral pH. It has a triblock structure denoted as C2S(H)48C2, which consists of hydrophilic collagen-inspired and histidine-rich silk-inspired blocks. The protein was successfully produced by the yeast Pichia pastoris in laboratory-scale bioreactors, and it was purified by selective precipitation. This efficient and inexpensive production method provided material of sufficient quantities, purity and sterility for cell culture study. Rheology and erosion studies showed that it forms hydrogels exhibiting long-term stability, self-healing behavior and tunable mechanical properties. Primary rat bone marrow cells cultured in direct contact with these hydrogels remained fully viable; however, proliferation and mineralization were relatively low compared to collagen hydrogel controls, probably because of the absence of cell-adhesive motifs. As biofunctional factors can be readily incorporated to improve material performance, our approach provides a promising route towards biomedical applications.

Synergistic stiffening in double-fiber networks.

Many biological materials are composite structures, interpenetrating networks of different types of fibers. The composite nature of such networks leads to superior mechanical properties, but the origin of this mechanical synergism is still poorly understood. Here we study soft composite networks, made by mixing two self-assembling fiber-forming components. We find that the elastic moduli of the composite networks significantly exceed the sum of the moduli of the two individual networks. This mechanical enhancement is in agreement with recent simulations, where it was attributed to a suppression of non-affine deformation modes in the most rigid fiber network due to the reaction forces in the softer network. The increase in affinity also causes a loss of strain hardening and an increase in the critical stress and stain at which the network fails.

Dilute self-healing hydrogels of silk-collagen-like block copolypeptides at neutral pH.

We report on self-healing, pH-responsive hydrogels that are entirely protein-based. The protein is a denovo designed recombinant triblock polypeptide of 66 kg/mol consisting of a silk-like middle block (GAGAGAGH)48, flanked by two long collagen-inspired hydrophilic random coil side blocks. The pH-dependent charge on the histidines in the silk block controls folding and stacking of the silk block. At low pH the protein exists as monomers, but above pH 6 it readily self-assembles into long fibers. At higher concentrations the fibers form self-healing physical gels. Optimal gel strength and self-healing are found at a pH of around 7. The modulus of a 2 wt % gel at pH 7 is G' = 1700 Pa. Being protein-based, and amenable to further sequence engineering, we expect that these proteins are promising scaffold materials to be developed for a broad range of biomedical applications.

Fibril formation by pH and temperature responsive silk-elastin block copolymers.

In this report, we study the self-assembly of two silk-elastin-like proteins: one is a diblock S(24)E(40) composed of 24 silk-like (S) repeats and 40 elastin-like (E) repeats; the other is a triblock S(12)C(4)E(40), in which the S and E blocks are separated by a random coil block (C(4)). Upon lowering the pH, the acidic silk-like blocks fold and self-assemble into fibrils by a nucleation-and-growth process. While silk-like polymers without elastin-like blocks form fibrils by heterogeneous nucleation, leading to monodisperse populations, the elastin-like blocks allow for homogeneous nucleation, which gives rise to polydisperse length distributions, as well as a concentration-dependent fibril length. Moreover, the elastin-like blocks introduce temperature sensitivity: at high temperature, the fibrils become sticky and tend to bundle and aggregate in an irreversible manner. Concentrated solutions of S(12)C(4)E(40) form weak gels at low pH that irreversibly lose elasticity in temperature cycling; this is also attributed to fibril aggregation.

Coating of single DNA molecules by genetically engineered protein diblock copolymers.

Coating DNA is an effective way to modulate its physical properties and interactions. Current chemosynthetic polymers form DNA aggregates with random size and shape. In this study, monodisperse protein diblock copolymers are produced at high yield in recombinant yeast. They carry a large hydrophilic colloidal block (≈400 amino acids) linked to a short binding block (≈12 basic amino acids). It is demonstrated that these protein polymers complex single DNA molecules as highly stable nanorods, reminiscent of cylindrical viruses. It is proposed that inter- and intramolecular bridging of DNA molecules are prevented completely by the small size of the binding block attached to the large colloidal stability block. These protein diblocks serve as a scaffold that can be tuned for application in DNA-based nanotechnology.

Tuning of collagen triple-helix stability in recombinant telechelic polymers.

The melting properties of various triblock copolymers with random coil middle blocks (100-800 amino acids) and triple helix-forming (Pro-Gly-Pro)(n) end blocks (n = 6-16) were compared. These gelatin-like molecules were produced as secreted proteins by recombinant yeast. The investigated series shows that the melting temperature (T(m)) can be genetically engineered to specific values within a very wide range by varying the length of the end block. Elongation of the end blocks also increased the stability of the helices under mechanical stress. The length-dependent melting free energy and T(m) of the (Pro-Gly-Pro)(n) helix appear to be comparable for these telechelic polymers and for free (Pro-Gly-Pro)(n) peptides. Accordingly, the T(m) of the polymers appeared to be tunable independently of the nature of the investigated non-cross-linking middle blocks. The flexibility of design and the amounts in which these nonanimal biopolymers can be produced (g/L range) create many possibilities for eventual medical application.

Self-assembly of silk-collagen-like triblock copolymers resembles a supramolecular living polymerization.

We produced several pH-responsive silk-collagen-like triblocks, one acidic and two alkaline. At pH values where the silk-like block is uncharged the triblocks self-assemble into filaments. The pH-induced self-assembly was examined by atomic force microscopy, light scattering, and circular dichroism. The populations of filaments were found to be very monodisperse, indicating that the filaments start to grow from already present nuclei in the sample. The growth then follows pseudo-first-order kinetics for all examined triblocks. When normalized to the initial concentration, the growth curves of each type of triblock overlap, showing that the self-assembly is a generic process for silk-collagen-silk triblocks, regardless of the nature of their chargeable groups. The elongation speed of the filaments is slow, due to the presence of repulsive collagen-like blocks and the limited number of possibilities for an approaching triblock to successfully attach to a growing end. The formation of filaments is fully reversible. Already present filaments can start growing again by addition of new triblocks. The structure of all filaments is very rich in ß-turns, leading to ß-rolls. The triblocks attain this structure only when attaching to a growing filament.