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1.
Biophys Rev (Melville) ; 4(3): 031301, 2023 Sep.
Article in English | MEDLINE | ID: mdl-38510706

ABSTRACT

Spider silk fibers are of scientific and industrial interest because of their extraordinary mechanical properties. These properties are normally determined by tensile tests, but the values obtained are dependent on the morphology of the fibers, the test conditions, and the methods by which stress and strain are calculated. Because of this, results from many studies are not directly comparable, which has led to widespread misconceptions in the field. Here, we critically review most of the reports from the past 50 years on spider silk mechanical performance and use artificial spider silk and native silks as models to highlight the effect that different experimental setups have on the fibers' mechanical properties. The results clearly illustrate the importance of carefully evaluating the tensile test methods when comparing the results from different studies. Finally, we suggest a protocol for how to perform tensile tests on silk and biobased fibers.

2.
Adv Funct Mater ; 32(23): 2200986, 2022 Jun 03.
Article in English | MEDLINE | ID: mdl-36505976

ABSTRACT

Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers' mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in ß-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high ß-strand propensity and can mediate tight inter-ß-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger ß-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L-1 which is in line with requirements for economically feasible bulk scale production.

3.
Sci Rep ; 12(1): 3507, 2022 03 03.
Article in English | MEDLINE | ID: mdl-35241705

ABSTRACT

Silk fibres attract great interest in materials science for their biological and mechanical properties. Hitherto, the mechanical properties of the silk fibres have been explored mainly by tensile tests, which provide information on their strength, Young's modulus, strain at break and toughness modulus. Several hypotheses have been based on these data, but the intrinsic and often overlooked variability of natural and artificial silk fibres makes it challenging to identify trends and correlations. In this work, we determined the mechanical properties of Bombyx mori cocoon and degummed silk, native spider silk, and artificial spider silk, and compared them with classical commercial carbon fibres using large sample sizes (from 10 to 100 fibres, in total 200 specimens per fibre type). The results confirm a substantial variability of the mechanical properties of silk fibres compared to commercial carbon fibres, as the relative standard deviation for strength and strain at break is 10-50%. Moreover, the variability does not decrease significantly when the number of tested fibres is increased, which was surprising considering the low variability frequently reported for silk fibres in the literature. Based on this, we prove that tensile testing of 10 fibres per type is representative of a silk fibre population. Finally, we show that the ideal shape of the stress-strain curve for spider silk, characterized by a pronounced exponential stiffening regime, occurs in only 25% of all tested spider silk fibres.


Subject(s)
Bombyx , Spiders , Animals , Carbon Fiber , Sample Size , Silk , Stress, Mechanical , Tensile Strength
4.
Materials (Basel) ; 15(3)2022 Jan 18.
Article in English | MEDLINE | ID: mdl-35160653

ABSTRACT

Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties. The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers. Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features. However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process. Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers. In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength. Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers.

5.
Microb Cell Fact ; 20(1): 150, 2021 Jul 30.
Article in English | MEDLINE | ID: mdl-34330289

ABSTRACT

BACKGROUND: The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for Escherichia coli. RESULTS: A fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures. CONCLUSIONS: The protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Batch Cell Culture Techniques/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Molecular Chaperones/genetics , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/metabolism , Humans , Molecular Chaperones/metabolism , Recombinant Proteins/metabolism
6.
Amyloid ; 28(3): 158-167, 2021 Sep.
Article in English | MEDLINE | ID: mdl-33583280

ABSTRACT

More than 30 proteins and peptides have been found to form amyloid fibrils in human diseases. Fibrils formed by transthyretin (TTR) are associated with ATTR amyloidosis, affecting many vital organs, including the heart and peripheral nervous system. Congo red staining is the gold standard method for detection of amyloid deposits in tissue. However, Congo red staining and amyloid typing methods such as immunofluorescence labelling are limited to relatively large deposits. Detection of small ATTR deposits present at an early stage of the disease could enable timely treatment and prevent severe tissue damage. In this study, we developed an enhanced ATTR amyloid detection method that uses functionalised protein nanofibrils. Using this method, we achieved sensitive detection of monomeric TTR in a microplate immunoassay and immunofluorescence labelling of ex vivo tissue from two patients containing ATTR aggregates. The system's utility was confirmed on sections from a patient with AA amyloidosis and liver sections from inflamed mouse. These results suggest that the detection system constitutes important new technology for highly sensitive detection of microscopic amounts of ATTR amyloid deposited in tissue.


Subject(s)
Amyloid Neuropathies, Familial , Amyloidosis , Amyloid , Amyloid Neuropathies, Familial/diagnosis , Amyloid Neuropathies, Familial/genetics , Amyloidogenic Proteins , Animals , Humans , Mice , Prealbumin/genetics , Serum Amyloid A Protein
7.
Molecules ; 25(14)2020 Jul 16.
Article in English | MEDLINE | ID: mdl-32708777

ABSTRACT

Efficient production of artificial spider silk fibers with properties that match its natural counterpart has still not been achieved. Recently, a biomimetic process for spinning recombinant spider silk proteins (spidroins) was presented, in which important molecular mechanisms involved in native spider silk spinning were recapitulated. However, drawbacks of these fibers included inferior mechanical properties and problems with low resistance to aqueous environments. In this work, we show that ≥5 h incubation of the fibers, in a collection bath of 500 mM NaAc and 200 mM NaCl, at pH 5 results in fibers that do not dissolve in water or phosphate buffered saline, which implies that the fibers can be used for applications that involve wet/humid conditions. Furthermore, incubation in the collection bath improved the strain at break and was associated with increased ß-sheet content, but did not affect the fiber morphology. In summary, we present a simple way to improve artificial spider silk fiber strain at break and resistance to aqueous solvents.


Subject(s)
Biomimetics , Fibroins/chemical synthesis , Recombinant Proteins/chemistry , Silk/chemistry , Animals , Fibroins/chemistry , Materials Testing , Recombinant Proteins/chemical synthesis , Recombinant Proteins/genetics , Solvents/chemistry , Spiders , Stress, Mechanical , Tensile Strength , Water/chemistry
8.
J Biol Chem ; 294(41): 14966-14977, 2019 10 11.
Article in English | MEDLINE | ID: mdl-31416835

ABSTRACT

Concerns over the environment are a central driver for designing cell-free enzymatic cascade reactions that synthesize non-petrol-based commodity compounds. An often-suggested strategy that would demonstrate the economic competitiveness of this technology is recycling of valuable enzymes through their immobilization. For this purpose, amyloid nanofibrils are an ideal scaffold to realize chemistry-free covalent enzyme immobilization on a material that offers a large surface area. However, in most instances, only single enzyme-functionalized amyloid fibrils have so far been studied. To embark on the next stage, here we displayed xylanase A, ß-xylosidase, and an aldose sugar dehydrogenase on Sup35(1-61) nanofibrils to convert beechwood xylan to xylonolactone. We characterized this enzymatic cascade by measuring the time-dependent accumulation of xylose, xylooligomers, and xylonolactone. Furthermore, we studied the effects of relative enzyme concentrations, pH, temperature, and agitation on product formation. Our investigations revealed that a modular cascade with a mixture of xylanase and ß-xylosidase, followed by product removal and separate oxidation of xylose with the aldose sugar dehydrogenase, is more productive than an enzyme mix containing all of these enzymes together. Moreover, we found that the nanofibril-coupled enzymes do not lose activity compared with their native state. These findings provide proof of concept of the feasibility of functionalized Sup35(1-61) fibrils as a molecular scaffold for biocatalytic cascades consisting of reusable enzymes that can be used in biotechnology.


Subject(s)
Amyloid/chemistry , Biocatalysis , Nanostructures/chemistry , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Protein Aggregates , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Biotechnology , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Kinetics , Models, Molecular , Oxidation-Reduction , Protein Structure, Secondary , Xylose/metabolism
9.
ACS Nano ; 12(9): 9363-9371, 2018 09 25.
Article in English | MEDLINE | ID: mdl-30207696

ABSTRACT

Amyloid nanofibrils are excellent scaffolds for designable materials that can be endowed with biotechnologically relevant functions. However, most of all excellent ideas and concepts that have been reported in the literature might never see real-world implementation in biotechnological applications. One bottleneck is the large-scale production of these materials. In this paper, we present an attempt to create a generic and scalable platform for producing ready-to-use functionalized nanofibrils directly from a eukaryotic organism. As a model material, we assembled Sup35(1-61) amyloid nanofibrils from Saccharomyces cerevisiae decorated with the Z-domain dimer, which has a high affinity toward antibody molecules. To this end, Komagataella pastoris was engineered by inserting gene copies of Sup35(1-61) and the protein chimera Sup35(1-61)-ZZ into the genome. This strain has the capability to constantly secrete amyloidogenic proteins into the extracellular medium, where the mature functionalized fibrils form, with a production yield of 35 mg/L culture. Another striking feature of this strategy is that the separation of the fibril material from the cells requires only centrifugation and resuspension in saline water. The fast production rates, minimal hands-on time, and high stability of the assembled material are some highlights that make the direct assembly of functionalized fibrils in the extracellular medium an alternative to production methods that are not suitable for large-scale production of designed amyloids.


Subject(s)
Nanofibers/chemistry , Peptide Termination Factors/biosynthesis , Pichia/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Models, Molecular , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Pichia/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
10.
PLoS One ; 13(4): e0196250, 2018.
Article in English | MEDLINE | ID: mdl-29684061

ABSTRACT

Enzymatic functionalization of cross-ß structured protein nanofibrils has hitherto resulted in a severe reduction of the catalytic efficiency of high turnover biocatalysts. It has been speculated that steric restrictions and mass transport pose limits on the attached enzymes, but detailed kinetics analyzing this have not yet been reported. For a more comprehensive understanding, we studied protein nanofibrils endowed with TEM1, a ß-lactamase from Escherichia coli. The packing density of TEM1 along the fibrils was controlled by co-fibrillation; in other words, the N-terminal ureidosuccinate transporter Ure2(1-80) from Saccharomyces cerevisiae was simultaneously aggregated with the chimeric proteins TEM1-Ure2(1-80). The mature fibrils were trapped in a column, and the rate of ampicillin hydrolysis was recorded using a continuous substrate flow. The turnover rate was plotted as a function of substrate molecules available per enzyme per second, which demonstrated that an elevated substrate availability counteracts mass transport limitations. To analyze this data set, we derived a kinetic model, which makes it possible to easily characterize and compare enzymes packed in columns. The functional TEM1 nanofibrils possess 80% of the catalytic turnover rate compared to free TEM1 in solution. Altogether, we have created protein nanofibrils that can effectively hydrolyze ß-lactam antibiotic contaminations and provided a groundwork strategy for other highly functional enzymatic nanofibrils.


Subject(s)
Anti-Bacterial Agents/pharmacokinetics , Bioreactors , Enzymes, Immobilized , Glutathione Peroxidase/metabolism , Nanofibers , Prions/metabolism , Saccharomyces cerevisiae Proteins/metabolism , beta-Lactamases/metabolism , Ampicillin/metabolism , Ampicillin/pharmacokinetics , Anti-Bacterial Agents/metabolism , Biocatalysis , Biodegradation, Environmental , Bioreactors/microbiology , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Escherichia coli/enzymology , Glutathione Peroxidase/chemistry , Hydrolysis , Kinetics , Nanofibers/chemistry , Prions/chemistry , Protein Multimerization , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/chemistry
11.
Sci Rep ; 7(1): 5949, 2017 07 20.
Article in English | MEDLINE | ID: mdl-28729665

ABSTRACT

Protofibrils of the 42 amino acids long amyloid-ß peptide are transient pre-fibrillar intermediates in the process of peptide aggregation into amyloid plaques and are thought to play a critical role in the pathology of Alzheimer's disease. Hence, there is a need for research reagents and potential diagnostic reagents for detection and imaging of such aggregates. Here we describe an in vitro selection of Affibody molecules that bind to protofibrils of Aß42cc, which is a stable engineered mimic of wild type Aß42 protofibrils. Several binders were identified that bind Aß42cc protofibrils with low nanomolar affinities, and which also recognize wild type Aß42 protofibrils. Dimeric head-to-tail fusion proteins with subnanomolar binding affinities, and very slow dissociation off-rates, were also constructed. A mapping of the chemical properties of the side chains onto the Affibody scaffold surface reveals three distinct adjacent surface areas of positively charged surface, nonpolar surface and a polar surface, which presumably match a corresponding surface epitope on the protofibrils. The results demonstrate that the engineered Aß42cc is a suitable antigen for directed evolution of affinity reagents with specificity for wild type Aß42 protofibrils.


Subject(s)
Amyloid beta-Peptides/metabolism , Peptide Fragments/metabolism , Protein Aggregates , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Cell Surface Display Techniques , Kinetics , Peptide Fragments/chemistry , Protein Binding , Protein Multimerization , Recombinant Fusion Proteins/chemistry
12.
Biotechnol J ; 12(6)2017 Jun.
Article in English | MEDLINE | ID: mdl-28371185

ABSTRACT

Elevated performance of instruments and electronic devices is frequently attained through miniaturization of the involved components, which increases the number of functional units in a given volume. Analogously, to conquer the limitations of materials used for the purification of monoclonal antibodies and for the sensitivity of immunoassays, the support for capturing antibodies requires miniaturization. A suitable scaffold for this purpose are cross-ß structured protein nanofibrils, as they offer a superior surface area over volume ratio and because manipulation can be implemented genetically. To display the antibody binding Z-domain dimers (ZZ) along the surface of the fibrils and grant maximal accessibility to the functional units, the N-terminal fragments of the fibrillating translation release factor Sup35 or ureidosuccinate transporter Ure2, both from Saccharomyces cerevisae, are simultaneously fibrillated with the chimeric-proteins Sup35-ZZ and ZZ-Ure2, respectively. Optimization of the fibril composition yields a binding capacity of 1.8 mg antibody per mg fibril, which is a binding capacity that is almost 20-fold higher, compared to the commercially available affinity medium gold standard, protein A sepharose. This study lifts the craft of nanofibril functionalization to the next level, and offers a universal framework to improve biomaterials that rely on the display of functional proteins or enzymes.


Subject(s)
Antibodies, Monoclonal/metabolism , Glutathione Peroxidase/metabolism , Peptide Termination Factors/metabolism , Prions/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Antibodies, Monoclonal/isolation & purification , Protein Binding , Protein Structure, Tertiary
13.
J Med Chem ; 57(5): 1802-11, 2014 Mar 13.
Article in English | MEDLINE | ID: mdl-24512311

ABSTRACT

To aid the design of next generation hepatitis C virus (HCV) drugs, the kinetics of the interactions between NS3 protease inhibitors and enzyme from genotypes 1a, 1b, and 3a have been characterized. The linear mechanism-based inhibitors VX-950 (telaprevir) and SCH 503034 (boceprevir) benefited from covalent adduct formation. However, the apparent affinities were rather weak (VX-950, K(D)* of 340, 8.5, and 1000 nM for genotypes 1a, 1b and 3a, respectively; SCH 503034, K(D)* of 90 and 3.9 nM for 1b and 3a, respectively). The non-mechanism-based macrocyclic inhibitors BILN-2016 (ciluprevir) and ITMN-191 (danoprevir) had faster association and slower dissociation kinetics, indicating that rigidification is kinetically favorable. ITMN-191 had nanomolar affinities for all genotypes (K(D)* of 0.13, 1.6, and 0.52 nM), suggesting that a broad spectrum drug is conceivable. The data show that macrocyclic scaffolds and mechanism-based inhibition are advantageous but that there is considerable room for improvement of the kinetics of HCV protease targeted drugs.


Subject(s)
Biosensing Techniques , Protease Inhibitors/pharmacology , Surface Plasmon Resonance/instrumentation , Viral Nonstructural Proteins/antagonists & inhibitors , Kinetics , Viral Nonstructural Proteins/genetics
14.
J Enzyme Inhib Med Chem ; 29(6): 868-76, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24517372

ABSTRACT

CONTEXT: Natural strain variation and rapid resistance development makes development of broad spectrum hepatitis C virus (HCV) drugs very challenging and evaluation of inhibitor selectivity and resistance must account for differences in the catalytic properties of enzyme variants. OBJECTIVE: To understand how to study selectivity and relationships between efficacy and genotype or resistant mutants for NS3 protease inhibitors. MATERIALS AND METHODS: The catalytic properties of NS3 protease from genotypes 1a, 1b and 3a, and their sensitivities to four structurally and mechanistically different NS3 protease inhibitors have been analysed under different experimental conditions. RESULTS: The optimisation of buffer conditions for each protease variant enabled the comparison of their catalytic properties and sensitivities to the inhibitors. All inhibitors were most effective against genotype 1a protease, with VX-950 having the broadest selectivity. DISCUSSION AND CONCLUSION: A new strategy for evaluation of inhibitors relevant for the discovery of broad spectrum HCV drugs was established.


Subject(s)
Antiviral Agents/chemistry , Drug Resistance, Viral/genetics , Genetic Variation , Hepacivirus/drug effects , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/genetics , Antiviral Agents/pharmacology , Carbamates/chemistry , Carbamates/pharmacology , Cloning, Molecular , Cyclopropanes , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genotype , Hepacivirus/enzymology , Hepacivirus/genetics , Isoindoles , Lactams/chemistry , Lactams/pharmacology , Lactams, Macrocyclic , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Mutation , Oligopeptides/chemistry , Oligopeptides/pharmacology , Proline/analogs & derivatives , Protease Inhibitors/pharmacology , Protein Structure, Tertiary , Quinolines/chemistry , Quinolines/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sulfonamides/chemistry , Sulfonamides/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry
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