Browsing by Author "Linder, Markus B."

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  • Advanced Materials through Assembly of Nanocelluloses
    (2018-06) Kontturi, Eero; Laaksonen, Päivi; Linder, Markus B.; Nonappa; Gröschel, André H.; Rojas, Orlando J.; Ikkala, Olli
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    There is an emerging quest for lightweight materials with excellent mechanical properties and economic production, while still being sustainable and functionalizable. They could form the basis of the future bioeconomy for energy and material efficiency. Cellulose has long been recognized as an abundant polymer. Modified celluloses were, in fact, among the first polymers used in technical applications; however, they were later replaced by petroleum-based synthetic polymers. Currently, there is a resurgence of interest to utilize renewable resources, where cellulose is foreseen to make again a major impact, this time in the development of advanced materials. This is because of its availability and properties, as well as economic and sustainable production. Among cellulose-based structures, cellulose nanofibrils and nanocrystals display nanoscale lateral dimensions and lengths ranging from nanometers to micrometers. Their excellent mechanical properties are, in part, due to their crystalline assembly via hydrogen bonds. Owing to their abundant surface hydroxyl groups, they can be easily modified with nanoparticles, (bio)polymers, inorganics, or nanocarbons to form functional fibers, films, bulk matter, and porous aerogels and foams. Here, some of the recent progress in the development of advanced materials within this rapidly growing field is reviewed.
  • Aligning cellulose nanofibril dispersions for tougher fibers
    (2017-12-01) Mohammadi, Pezhman; Toivonen, Matti S.; Ikkala, Olli; Wagermaier, Wolfgang; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Nanocomposite materials made from cellulose show a great potential as future high-performance and sustainable materials. We show how high aspect ratio cellulose nanofibrils can be efficiently aligned in extrusion to fibers, leading to increased modulus of toughness (area under the stress-strain curve), Young's modulus, and yield strength by increasing the extrusion capillary length, decreasing its diameter, and increasing the flow rate. The materials showed significant property combinations, manifesting as high modulus of toughness (~28-31 MJ/m3) vs. high stiffness (~19-20 GPa), and vs. high yield strength (~130-150 MPa). Wide angle X-ray scattering confirmed that the enhanced mechanical properties directly correlated with increased alignment. The achieved moduli of toughness are approximately double or more when compared to values reported in the literature for corresponding strength and stiffness. Our results highlight a possibly general pathway that can be integrated to gel-spinning process, suggesting the hypothesis that that high stiffness, strength and toughness can be achieved simultaneously, if the alignment is induced while the CNF are in the free-flowing state during the extrusion step by shear at relatively low concentration and in pure water, after which they can be coagulated.
  • Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation
    (2020-11-15) Fedorov, Dmitrii; Batys, Piotr; Hayes, David B.; Sammalkorpi, Maria; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the oligomerization behavior of CBMCipA, as such solution association may have an impact on its use. As the principal approach, we used sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data by a discrete model genetic algorithm and by building a binding isotherm based on weight average sedimentation coefficient and by global fitting of sedimentation equilibrium data we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of 90 ± 30 μM. As the KD of CBMCipA binding to cellulose is below 1 μM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at high concentrations used in some applications of the CBMCipA, its dimerization is likely to have a marked effect on its solution behavior.
  • Binding Forces of Cellulose Binding Modules on Cellulosic Nanomaterials
    (2019-02-11) Griffo, Alessandra; Rooijakkers, Bart J.M.; Hähl, Hendrik; Jacobs, Karin; Linder, Markus B.; Laaksonen, Päivi
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    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.
  • Binding of cellulose binding modules reveal differences between cellulose substrates
    (2016-10-17) Arola, Suvi; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    The interaction between cellulase enzymes and their substrates is of central importance to several technological and scientific challenges. Here we report that the binding of cellulose binding modules (CBM) from Trichoderma reesei cellulases Cel6A and Cel7A show a major difference in how they interact with substrates originating from wood compared to bacterial cellulose. We found that the CBM from TrCel7A recognizes the two substrates differently and as a consequence shows an unexpected way of binding. We show that the substrate has a large impact on the exchange rate of the studied CBM, and moreover, CBM-TrCel7A seems to have an additional mode of binding on wood derived cellulose but not on cellulose originating from bacterial source. This mode is not seen in double CBM (DCBM) constructs comprising both CBM-TrCel7A and CBM-TrCel6A. The linker length of DCBMs affects the binding properties, and slows down the exchange rates of the proteins and thus, can be used to analyze the differences between the single CBM. These results have impact on the cellulase research and offer new understanding on how these industrially relevant enzymes act.
  • Biological activity of multicomponent bio-hydrogels loaded with tragacanth gum
    (2022-08-31) Teixeira Polez, Roberta; Morits, Maria; Jonkergouw, Christopher; Phiri, Josphat; Valle-Delgado, Juan José; Seppälä, Jukka; Linder, Markus B.; Maloney, Thaddeus; Rojas Gaona, Orlando; Österberg, Monika
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Producing hydrogels capable of mimicking the biomechanics of soft tissue remains a challenge. We explore the potential of plant-based hydrogels as polysaccharide tragacanth gum and antioxidant lignin nanoparticles in bioactive multicomponent hydrogels for tissue engineering. These natural components are combined with TEMPO-oxidized cellulose nanofibrils, a material with known shear thinning behavior. Hydrogels presented tragacanth gum (TG) concentration-dependent rheological properties suitable for extrusion 3D printing. TG enhanced the swelling capacity up to 645% and the degradation rate up to 1.3%/day for hydrogels containing 75% of TG. Young's moduli of the hydrogels varied from 5.0 to 11.6 kPa and were comparable to soft tissues like skin and muscle. In vitro cell viability assays revealed that the scaffolds were non-toxic and promoted proliferation of hepatocellular carcinoma HepG2 cells. Therefore, the plant-based hydrogels designed in this work have a significant potential for tissue engineering.
  • Biomimetic composites with enhanced toughening using silk-inspired triblock proteins and aligned nanocellulose reinforcements
    (2019-09-13) Mohammadi, Pezhman; Sesilja Aranko, A.; Landowski, Christopher P.; Ikkala, Olli; Jaudzems, Kristaps; Wagermaier, Wolfgang; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Silk and cellulose are biopolymers that show strong potential as future sustainable materials. They also have complementary properties, suitable for combination in composite materials where cellulose would form the reinforcing component and silk the tough matrix. A major challenge concerns balancing structure and functional properties in the assembly process. We used recombinant proteins with triblock architecture, combining structurally modified spider silk with terminal cellulose affinity modules. Flow alignment of cellulose nanofibrils and triblock protein allowed continuous fiber production. Protein assembly involved phase separation into concentrated coacervates, with subsequent conformational switching from disordered structures into b sheets. This process gave the matrix a tough adhesiveness, forming a new composite material with high strength and stiffness combined with increased toughness. We show that versatile design possibilities in protein engineering enable new fully biological materials and emphasize the key role of controlled assembly at multiple length scales for realization.
  • Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins
    (2023-03-06) Fan, Ruxia; Hakanpää, Johanna; Elfving, Karoliina; Taberman, Helena; Linder, Markus B.; Aranko, A. Sesilja
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    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.
  • Bursting of condensates
    (2024-05-17) Tunn, Isabell; Beaune, Grégory; Tersteegen, Jennifer; Välisalmi, Teemu; Timonen, Jaakko V.I.; Brochard-Wyart, Françoise; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Numerous biomolecular shell-forming condensates are reported in cells and bioengineered in vitro. The relationship between the molecular structure of shell-forming condensates and their biophysical properties remains largely unexplored. To fill this gap, we characterize shell-forming condensates of bioengineered spider silk proteins based on Araneus diadematus major ampulla gland silk fibroin 3 (ADF3) using micropipette aspiration. We observe that condensates can burst during aspiration like soap bubbles or polymersomes, demonstrating the formation of a dense protein layer (shell) at the condensate interface. The tendency to burst is more pronounced for condensates formed from proteins with weakly dimerizing terminal blocks. We develop a model to analyse the aspiration and bursting of the condensates, to obtain the surface and bulk viscosity, and to estimate the shell thickness and viscosity. Understanding and controlling the bursting of condensates will open avenues for their use in materials, as compartments for reactions or drug delivery systems.
  • Coacervation of resilin fusion proteins containing terminal functionalities
    (2018-11-01) Fang, Wenwen; Nonappa; Vitikainen, Marika; Mohammadi, Pezhman; Koskela, Salla; Soikkeli, Miika; Westerholm-Parvinen, Ann; Landowski, Christopher P.; Penttilä, Merja; Linder, Markus B.; Laaksonen, Päivi
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Liquid-liquid phase transition known as coacervation of resilin-like-peptide fusion proteins containing different terminal domains were investigated. Two different modular proteins were designed and produced and their behavior were compared to a resilin-like-peptide without terminal domains. The size of the particle-like coacervates was modulated by the protein concentration, pH and temperature. The morphology and three-dimensional (3D) structural details of the coacervate particles were investigated by cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET) reconstruction. Selective adhesion of the coacervates on cellulose and graphene surfaces was demonstrated.
  • Controllable coacervation of recombinantly produced spider silk protein using kosmotropic salts
    (2020-02-15) Mohammadi, Pezhman; Christopher, Jonkergouw; Beaune, Grégory; Engelhardt, Peter; Kamada, Ayaka; Timonen, Jaakko V.I.; Knowles, Tuomas P.J.; Penttila, Merja; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Recent developments suggest that the phase transition of natural and synthetic biomacromolecules represents an important and ubiquitous mechanism underlying structural assemblies toward the fabrication of high-performance materials. Such a transition results in the formation of condensed liquid droplets, described as condensates or coacervates. Being able to effectively control the assembly of such entities is essential for tuning the quality and their functionality. Here we describe how self-coacervation of genetically engineered spidroin-inspired proteins can be preceded by a wide range of kosmotropic salts. We studied the kinetics and mechanisms of coacervation in different conditions, from direct observation of initial phase separation to the early stage of nucleation/growth and fusion into large fluid assemblies. We found that coacervation induced by kosmotropic salts follows the classical nucleation theory and critically relies on precursor clusters of few weak-interacting protein monomers. Depending on solution conditions and the strength of the supramolecular interaction as a function of time, coacervates with a continuum of physiochemical properties were observed. We observed similar characteristics in other protein-based coacervates, which include having a spherical-ellipsoid shape in solution, an interconnected bicontinuous network, surface adhesion, and wetting properties. Finally, we demonstrated the use of salt-induced self-coacervates of spidroin-inspired protein as a cellulosic binder in dried condition.
  • Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions
    (2024-07-17) Leppert, Axel; Feng, Jianhui; Railaite, Vaida; Bohn Pessatti, Tomas; Cerrato, Carmine P.; Mörman, Cecilia; Osterholz, Hannah; Lane, David P.; Maia, Filipe R.N.C.; Linder, Markus B.; Rising, Anna; Landreh, Michael
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    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.
  • Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels
    (2020-07) Rooijakkers, Bart J.M.; Arola, Suvi; Velagapudi, Rama; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.
  • Effect of oxidation on cellulose and water structure : a molecular dynamics simulation study
    (2021-05) Mudedla, Sathish Kumar; Vuorte, Maisa; Veijola, Elias; Marjamaa, Kaisa; Koivula, Anu; Linder, Markus B.; Arola, Suvi; Sammalkorpi, Maria
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Enzymatic cleavage of glycocidic bonds is an important, green and biocompatible means to refine lignocellulosic biomass. Here, the effect of the resulting oxidation point defects on the structural and water interactions of crystalline cellulose {100} surface are explored using classical molecular dynamics simulations. We show that even single oxidations reduce the connections within cellulose crystal significantly, mostly via local interactions between the chains along the surface plane but also via the oxidation defects changing the structure of the crystal in direction perpendicular to the surface. Hydrogen bonding on the surface plane of cellulose is analyzed to identify onset of desorption of glucose chains, and the desorption probed. To assess the actual soluble product profile and their fractions resulting from lytic polysaccharide monooxygenase (LPMO) enzyme oxidation on real cellulose crystal samples, we employ High-Performance Anion-Exchange Chromatography with Pulsed Amperometric-Detection (HPAEC-PAD) technique. The findings demonstrate the LPMO oxidation results in soluble glucose fragments ranging from 2 to 8 glucose units in length. Additionally, significantly more oxidized oligosaccharides were released in LPMO treatment of AaltoCell than Avicel, the two studied microcrystalline cellulose species. This is likely to result from the large reactive surface area preserved in AaltoCell due to manufacturing process. Furthermore, as can be expected, the oxidation defects at the surfaces lead to the surfaces binding a larger amount of water both via direct influence by the defect but also the defect induced protrusions and fluctuations of the glucose chain. We quantify the enhancement of water interactions of cellulose crystals due to the oxidation defects, even when no desorption takes place. The molecular simulations indicate that the effect is most pronounced for the C1-acid oxidation (carboxylic acid formation) but present also for the other defects resulting from oxidation. The findings bear significance in understanding the effects of enzymatic oxidation on cellulose nanocrystals, the difference between cellulose species, and cleavage of soluble products from the cellulosic material.
  • Effect of Phosphate on the Molecular Properties, Interactions, and Assembly of Engineered Spider Silk Proteins
    (2024-07-08) Yin, Yin; Griffo, Alessandra; Gutiérrez Cruz, Adrián; Hähl, Hendrik; Jacobs, Karin; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    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 β-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 β-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 β-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.
  • Electrolyte-Controlled Permeability in Nanocellulose-Stabilized Emulsions
    (2022-09-13) Heise, Katja; Jonkergouw, Christopher; Anaya-Plaza, Eduardo; Guccini, Valentina; Pääkkönen, Timo; Linder, Markus B.; Kontturi, Eero; Kostiainen, Mauri A.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Particle-stabilized emulsions, so-called Pickering emulsions (PEs), are a promising low-tech avenue to precisely engineered materials for applications in drug delivery, catalysis, or water remediation. The particle assembly at the liquid–liquid interface provides superior stability and an adjustable permeability, which is a key parameter for controllable compound capture and release. However, understanding the complex factors that control the particle assembly in detail is a still-remaining challenge limiting practical applications of PEs in an industrial framework. In this study, the properties of oil-in-water emulsions, stabilized by cellulose nanocrystals (CNCs), are investigated. It is shown how high ionic strength leads to low polydispersity droplets, with restricted permeability across the oil-water interface due to the dense packing of the CNC layer. In contrast, lower electrolyte concentration enables enhanced uptake through the interface, while providing the required stability for the reusability of the material. The authors continue to study the impact of the electrolyte content on the dynamic responses of the emulsions, leading to a liquid–liquid system with tunable cyclic uptake and release levels. Overall, the results highlight the potential of nanocellulose-stabilized emulsions as tunable and robust material platform with well-defined permeability characteristics—made in a simple way.
  • Emergence of Elastic Properties in a Minimalist Resilin-Derived Heptapeptide upon Bromination
    (2022-08) Pizzi, Andrea; Sori, Lorenzo; Pigliacelli, Claudia; Gautieri, Alfonso; Andolina, Clara; Bergamaschi, Greta; Gori, Alessandro; Panine, Pierre; Grande, Antonio Mattia; Linder, Markus B.; Baldelli Bombelli, Francesca; Soncini, Monica; Metrangolo, Pierangelo
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    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 β-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.
  • Exploration of Chemical Diversity in Intercellular Quorum Sensing Signalling Systems in Prokaryotes
    (2024-01-08) Jonkergouw, Christopher; Savola, Pihla; Osmekhina, Ekaterina; van Strien, Joeri; Batys, Piotr; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    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.
  • Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin
    (2018-05-01) Rooijakkers, Bart J.M.; Ikonen, Martina S.; Linder, Markus B.
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain’s sequence–function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.
  • Graphene Biosensor Programming with Genetically Engineered Fusion Protein Monolayers
    (2016-03-30) Soikkeli, Miika; Kurppa, Katri; Kainlauri, Markku; Arpiainen, Sanna; Paananen, Arja; Gunnarsson, David; Joensuu, Jussi J.; Laaksonen, Päivi; Prunnila, Mika; Linder, Markus B.; Ahopelto, Jouni
     A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
    We demonstrate a label-free biosensor concept based on specific receptor modules, which provide immobilization and selectivity to the desired analyte molecules, and on charge sensing with a graphene field effect transistor. The receptor modules are fusion proteins in which small hydrophobin proteins act as the anchor to immobilize the receptor moiety. The functionalization of the graphene sensor is a single-step process based on directed self-assembly of the receptor modules on a hydrophobic surface. The modules are produced separately in fungi or plants and purified before use. The modules form a dense and well-oriented monolayer on the graphene transistor channel and the receptor module monolayer can be removed, and a new module monolayer with a different selectivity can be assembled in situ. The receptor module monolayers survive drying, showing that the functionalized devices can be stored and have a reasonable shelf life. The sensor is tested with small charged peptides and large immunoglobulin molecules. The measured sensitivities are in the femtomolar range, and the response is relatively fast, of the order of one second. (Graph Presented).