Development of an artificial silk protein on the basis of a lacewing egg stalk protein

Development of an artificial silk protein on the basis of a lacewing egg stalk protein Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften An der Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften der Universität Bayreuth vorgelegt von Diplom Biologe Felix Bauer Bayreuth, Mai 2013 Die vorliegende Arbeit wurde in der Zeit von Juli / 2008 bis November /2013 in Bayreuth am Lehrstuhl Biomaterialien unter Betreuung von Herrn Professor Dr. Thomas Scheibel angefertigt. Vollständiger Abdruck der von der Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften (BayNAT) der Universität Bayreuth genemigten Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr.rer. nat.) Dissertation eingereicht am: 17.05.2013 Zulassung durch das Leitungsgremium: 10.06.2013 Wissenschaftliches Kolloquium: 14.11.2013 Amtierender Direktor: Prof. Dr. Franz Xaver Schmid Prüfungsausschuss: Prof. Dr. Thomas Scheibel (Erstgutachter) PD Dr. Stefan Geimer (Zweitgutachter) Prof. Dr. Andreas Fery (Vorsitz) Prof. Dr. Birgitta Wöhrl Content I CONTENT 1. SUMMARY ................................................................................................................. 1 2. ZUSAMMENFASSUNG ................................................................................................ 3 3. INTRODUCTION.......................................................................................................... 7 Silk 7 3.1. 3.1.1. Structure ........................................................................................................................................... 8 3.1.2. Myriapoda silk ................................................................................................................................... 9 3.1.3. Spider silk ........................................................................................................................................ 10 3.1.4. Insect silk ......................................................................................................................................... 11 3.1.4.1. Caddisfly silk ........................................................................................................................... 12 3.1.4.2. Lacewing silk ........................................................................................................................... 13 3.1.4.2.1. Cocoon silk ........................................................................................................................... 14 3.1.4.2.2. Egg stalk silk ......................................................................................................................... 14 3.1.4.2.3. Production of lacewing egg stalks ........................................................................................ 15 3.1.4.2.4. The colleterial gland ............................................................................................................. 17 3.1.4.2.5. Protein sequences/dope composition ................................................................................. 18 3.1.4.2.6. Mechanics of lacewing egg stalks ........................................................................................ 18 3.1.4.2.7. Structure .............................................................................................................................. 19 Recombinant production of silk proteins 20 3.2. Technical processing of silk proteins 21 3.3. Aims of the work 24 3.4. 4. OVERVIEW OF THE THESIS INCLUDING UNPUBLISHED DATA ..................................... 25 Mechanical analysis of natural lacewing egg stalks and fibres of caddisflies 25 4.1. Structural analysis of lacewing egg stalk silk 26 4.2. Silk gland analysis of lacewings 28 4.3. Biotechnological production of N[AS]8C, an artificial lacewing egg stalk protein 30 4.4. Fibre/stalk formation and analysis 33 4.5. Further processing of a recombinant lacewing protein 37 4.6. 4.6.1. Films ................................................................................................................................................ 37 4.6.2. Capsules .......................................................................................................................................... 39 Content 4.6.3. Hydrogels and foams ....................................................................................................................... 40 Cell culture on structured films 42 4.7. Individual contributions to joined publications 46 4.8. 5. LITERATURE ............................................................................................................. 47 6. LIST OF ABBREVIATIONS ........................................................................................... 63 7. DEPENDENCE OF MECHANICAL PROPERTIES OF LACEWING EGG STALKS ON RELATIVE HUMIDITY ....................................................................................................................... 65 8. ARTIFICIAL EGG STALKS MADE OF A RECOMBINANTLY PRODUCED SILK PROTEIN ..... 75 9. CONTROLLABLE CELL ADHESION, GROWTH AND ORIENTATION ON LAYERED SILK PROTEIN FILMS ............................................................................................................... 83 10. LIST OF PUBLICATIONS AND PATENTS....................................................................... 91 11. ACKNOWLEDGEMENT .............................................................................................. 93 12. ERKLÄRUNG: ............................................................................................................ 95 Summary 1 1. Summary Silks are widely used in textile industry as clothing and furnishings due to their tensile strength, smoothness, soft texture, lustre, and drape. Most commonly silk of the mulberry silkworm Bombyx mori (B. mori) is used in such applications, however, silks evolved independently in many different arthropods for various purposes.1 During evolution the different silks were optimised for their task-specific uses over millions of years, e.g. adopting different mechanical properties. The mechanical properties mainly derive from the protein secondary structure and its higher order arrangement in silk fibres. Spider silk, for example, is known for its tensile properties surpassing nylon, Kevlar®, silkworm silk, and high-tensile steel.2-5 Beyond their mechanical properties, some silks are also reported to be biocompatible and non-immunogenic.6 One beneficial feature of silk proteins is the possibility to process them into various morphologies.7, 8 Several of these silk features make them interesting for material scientists, intending to produce silks with tuneable properties depending on the desired application, ranging from technical ones such as high performance fibres to medical ones such as drug delivery. This thesis deals with the characterisation and reproduction of a less explored silk, the lacewing egg stalk silk. Mechanical testing revealed a strong dependence on the relative humidity. In the dry state at 30% relative humidity, the stalks are quite rigid and break at an elongation of 2% whereas at 70% and 100% relative humidity they elongate up to 434%. This extension is accompanied by a secondary structure change from crossß to parallel-ß. The cross-ß structure in unstretched stalks provides bending stiffness and rigidity to the stalk, and this bending stiffness gets lost when the stalks are stretched. In this thesis a model is proposed which explains these differences at various relative humidity on the molecular level, wherein changes in the strength of hydrogen bonds upon exposure to water (a hydrogen bond donor/acceptor) in combination with multiple disulphide cross-links (which are not affected by water) act together and are responsible for this behaviour. Summary 2 Based on consensus sequences of published sequence data (derived from MalXB2 an egg stalk protein of Mallada signata (M. signata)),9 an engineered egg stalk protein named N[AS]8C was recombinantly produced. To produce an artificial stalk, a droplet of a solution of purified N[AS]8C was placed on a substrate, and tweezers were used to pull out a fibre. After drying, and post treatment, the properties of the artificial stalks were investigated in comparison to the natural ones. Mechanical testing revealed similar behaviour at 30% relative humidity, but at 70% and 100% relative humidity the artificial stalks were not as extensible as the natural ones. This corresponds to the fact, that no cross-ß structure was formed, and, therefore, no rearrangement into parallel-ß structure was possible. Subsequently, N[AS]8C was processed into non-fibrous morphologies. It was possible to produce capsules, hydrogels, foams, and films. The foams show an interesting micro and nano structure which differs from that of recombinant spider silk. The cavities are filled with a mesh of nano fibres building a 3D scaffold. Films are a morphology with potential for application in cell culture. Fibroblast attachment on N[AS]8C films is quite poor. Therefore, we tried to induce guided fibroblast growth on patterned protein films. A first layer of the films was cast from ntagCysC16-c(RGDfK), an engineered spider silk protein coupled with the integrin recognition motif RGD to provide a protein layer to which fibroblasts attached well. The second protein layer was produced using a PDMS (polydimethylsiloxane) template and N[AS]8C. Fibroblasts grown on these films adhere only to the RGD modified spider silk and not to the N[AS]8C areas. A second feature of such films is to orient the fibroblasts on films with alternating lines of the two proteins. Such films might be useful for tissue engineering to control cell adhesion and get a structured cell pattern. This is essential for many tissues such as bones, muscles, and epithelia tissue. The low cell adhesion properties of N[AS]8C films might be interesting for coatings for applications where cell adhesion is not desired such as stents or catheters. Zusammenfassung 3 2. Zusammenfassung Seide ist wegen ihrer Reißfestigkeit, Glätte, weichen Textur, ihres Glanzes und ihres Faltenwurfs ein in der Textilindustrie weit verbreitetes Material für Kleidungsstücke und Einrichtungsgegenstände. Meist wird für solche Anwendungen die Seide des Maulbeerspinners Bombyx mori (B. mori) verwendet. Seiden entwickelten sich unabhängig in vielen Arthropoden und werden zu verschiedensten Zwecken verwendet.1 Für diese wurden sie seit Millionen von Jahren durch die Evolution optimiert. Zum Beispiel haben Seiden unterschiedliche mechanische Eigenschaften entwickelt, welche hauptsächlich von der Sekundärstruktur der Proteine und ihrer übergeordneten Anordnung in den Seidenfäden abhängen. Spinnenseide ist beispielsweise bekannt für ihre Zugdehnungseigenschaften, welche die von Nylon, Kevlar®, Seidenspinnerseide und hochfestem Stahl überragen.2-5 Darüber hinaus gelten viele Seiden als biokompatibel und nicht immunogen.6 Eine weitere nützliche Eigenschaft von Seidenproteinen ist deren Verarbeitbarkeit in viele verschiedene Morphologien.7, 8 Viele dieser Eigenschaften machen Seiden interessant für Materialforscher, welche Seide mit gezielt beeinflussbaren Eigenschaften, abhängig von der erwünschten Anwendung, produzieren wollen. Denkbare Anwendungen reichen von Hochleistungsfasern für technische Anwendungen bis zu medizinischen Anwendungen, wie gezielte Pharmakotherapie. Diese Dissertation beschäftigt sich mit der Charakterisierung und der rekombinanten Herstellung eines wenig erforschten Seidenproteins aus Florfliegen Eierstielen. Mechanische Tests an Florfliegen Eierstielen zeigten eine starke Abhängigkeit der Dehnbarkeit von der Luftfeuchtigkeit. Im trockenen Zustand bei 30% relativer Luftfeuchtigkeit sind die Stiele biegesteif und brechen bei einer Dehnung von 2%, wohingegen sie bei 70% und 100% relativer Luftfeuchtigkeit bis zu ca. 430% dehnbar sind. Diese Dehnung korreliert mit einer Sekundärstrukturänderung von cross-ß zu parallel-ß. Die cross-ß Struktur in ungestreckten Stielen wird für deren Biegesteifigkeit verantwortlich gemacht. Diese Biegesteifigkeit geht verloren, wenn die Stiele gestreckt werden. In dieser Arbeit wird ein Modell vorgeschlagen, welches die Unterschiede bei verschiedener Luftfeuchtigkeit auf molekularer Ebene erklärt. Verantwortlich für dieses Verhalten sind Änderungen in der Bindungsenergie von Zusammenfassung 4 Wasserstoffbrückenbindungen durch die Anwesenheit von Wasser (Wasserstoffbrücken Donor/Akzeptor) im Zusammenspiel mit Disulfidbindungen, die nicht durch das Wasser beeinflusst werden. Basierend auf Konsensussequenzen des veröffentlichten Eierstiel Proteins MalXB2 wurde das Protein N[AS]8C, eine künstlich konstruierte Variante des Proteins, biotechnologisch hergestellt.9 Um einen künstlichen Stiel zu produzieren wurde ein Tropfen einer Lösung von gereinigtem N[AS]8C auf einen Untergrund aufgebracht und anschließend aus dem Tropfen mithilfe einer Pinzette Fäden gezogen. Nach dem Trocknen und Nachbehandeln der Fäden wurden die Eigenschaften der künstlichen sowie der natürlichen Stiele untersucht. Mechanische Analysen zeigten ähnliche Eigenschaften der natürlichen und künstlichen Eierstiele bei 30% relativer Luftfeuchtigkeit, wohingegen die künstlichen Stiele bei 70% und 100% weniger dehnbar waren als die natürlichen. Dies stimmt mit der Tatsache überein, dass in den künstlichen Stielen keine cross-ß Struktur gebildet wurde und somit keine Umformung von cross-ß zu parallel-ß stattfinden konnte. In einem weiteren Schritt wurde N[AS]8C in weitere Morphologien verarbeitet. Es war möglich Kapseln, Hydrogele, Schäume und Filme herzustellen. Die Schäume zeigen eine interessante Mikround Nano-Strukturierung, die sich von Spinnenseiden-Schäumen unterscheidet. Die Poren sind von Nano-Fasern durchzogen, die ein 3D Netzwerk bilden. Diese Schäume könnten für weitere Untersuchungen und Anwendungen als Filtermaterial oder Zellkultur-Gerüst interessant sein. Eine Morphologie mit Potential zur Anwendung in der Zellkultur sind Filme. Da Fibroblasten schlecht auf Filmen aus N[AS]8C adhärieren, wurde versucht, Fibroblasten gezielt auf einem gemusterten Film wachsen zu lassen. Die Grundschicht wurde aus ntagCysC16-c(RGDfK), einer technisch erzeugten Variante eines Spinnenseidenproteins, an welches die Integrin-Erkennungssequenz RGD gekoppelt ist, gegossen. Aufgrund dieser Erkennungssequenz adhärieren Fibroblasten gut auf diesen Filmen. Eine zweite Schicht aus N[AS]8C wurde mithilfe einer PDMS (Polydimethylsiloxan) Maske aufgebracht. Fibroblasten adhärieren auf solchen gemusterten Filmen nur auf den ntagCysC16-c(RGDfK) und nicht auf den mit N[AS]8C bedeckten Bereichen. Eine weitere Besonderheit dieser Filme ist die Zusammenfassung 5 Möglichkeit, durch Applikation des zweiten Films in Streifenform, Fibroblasten ausgerichtet wachsen zu lassen. Solche Filme könnten für „Tissue engineering“ genutzt werden, um Zelladhäsion zu kontrollieren und eine strukturierte Zellausrichtung zu erhalten. Dies ist essentiell für viele natürliche Gewebe wie Knochen, Muskeln, und Epithelgewebe. Des Weiteren könnten die schwachen Zelladhäsions-Eigenschaften von N[AS]8C Filmen für Beschichtungen, bei denen Zelladhäsion nicht erwünscht ist, wie beispielsweise Stents oder Kathetern, interessant sein. Introduction 12 helical structure, and these silks are used for building cocoons.21, 28, 49 The second type is a cross-ß silk, which is used in egg stalks and is secreted by the colleterial gland. 3.1.4.1. Caddisfly silk Caddisfly larvae are commonly used by ecologists to investigate the water quality of rivers and lakes. They are listed in the insect order Trichoptera and are Holometabola, which means they undergo metamorphosis with larvae and adults showing a totally different habitus. The adult flies attach their eggs under water or close above the water surface. The larvae depend on water as habitats where they undergo five to seven larval stages. The larvae feed on algae, leafs, and small water insects, depending on their species and are divided into two groups the eruciform (casemaking) and the campodeiform (free-living and net spinning) larvae (see Figure 2).78 The erudiform larvae build round cases by gluing together various materials such as stones, sand, or small organic material by the use of a silken thread. The net spinning campodeiform larvae use silk to build small nets to catch small water insects and build a silken retreat to hide from predators (see Figure 2).78 Larvae of both groups use silk to pupate in a cocoon.79 Caddisfly silk is produced in the labial glands which are homologue to the labial glands of butterflies and therefore the silk morphology and composition is comparable to lepidopteran silk. Two flattened ribbon like fibres are glued together by sericin-like glue.80 The silk is composed of homologues of heavy chain and light chain fibroins but no P25 was detected so far.20, 81 The caddisfly heavy chain fibroin has some differences to those of Lepidoptera, showing a high content of bulky and basic amino acid residues, a low alanine content, and a repeating motif containing phosphorylated serines which could provide crosslinking by Ca2+-ions.82 An additional protein, Nf-1, with a high content of cysteine is thought to crosslink the proteins by disulphide bonds, providing water insolubility to the fibres.83, 84 Introduction 13 Figure 2: A and B: Caddisfly cases built from little stones; C and D: Caddisfly nets spun in an artificial surrounding. Scale bars: 0.7 cm. 3.1.4.2. Lacewing silk Green lacewing larvae are known for eating aphids which they are bred for commercially. Lacewings such as Chrysopa carnea (C. carnea) (Neuroptera: Chrysopidae) are Holometabola. Normally insects are known to produce only one silk type, but lacewings produce two.9 The larvae (Figure 3 C) produce a cocoon before metamorphosis (Figure 3 D), while adult females (Figure 3 A) use a second type for a silken stalk to protect their eggs from predators (Figure 3 B).85-87 Introduction 14 Figure 3: Lifecycle of lacewings comprising an adult lacewing (A), eggs on stalks (B), larva (C), and pupated larva (D). 3.1.4.2.1. Cocoon silk The cocoon of lacewing larvae comprises two layers.21, 28, 88 First the larva secretes a silken thread from the malphigian tubules and deposits a loosely woven cocoon. Later the larva deposits an inner layer of lipids to protect itself from water loss during metamorphosis. The silken threads have diameters of about 2 µm, are composed of one protein which is 49 kDa in size, and are rich in alanine.21 The protein adopts an α-helical structure but surprisingly it does not assemble into coiled coils as other α-helical silks do.21 3.1.4.2.2. Egg stalk silk Female lacewings attach their eggs to silken stalks to protect the eggs from predators such as ants or their own larvae.87 The stalk is drawn from a secreted protein droplet produced in the colleterial gland. An amino acid composition analysis revealed high levels of serine (41%), glycine (24%) and alanine (20%).89 Investigations Introduction 15 of a c-DNA library of the lacewing (Mallada signata (M. signata)) colleterial gland yielded clones encoding two proteins: MalXB1 (86 kDa) and MalXB2 (55 kDa), both containing a highly repetitive central structure (over 70%) which has a 16 amino acid periodicity.9 It was proposed that eight amino acid long ß-strands fold into a regular antiparallel cross-ß structure which is stacked in fibreand side chain-direction. This structure converts into a parallel-ß structure upon stretching.47, 90 Further the stalks of some lacewing species such as Ceraeochrysa smithi are reported to be coated by droplets containing fatty acids, an ester, and various straight-chain aldehydes. This fluid was shown to protect the eggs from predators such as ants.86 3.1.4.2.3. Production of lacewing egg stalks The production of egg stalks seems to be quite simple compared to the complex spinning mechanisms of spiders where shear forces, pH change, and ion exchange play important roles.54, 91-100 The female lacewing first taps her abdomen a few times on the surface (Figure 4 A-C), deposits a droplet of spinning solution from the colleterial gland on a surface (Figure 4 D), dips the end of an egg into the solution, and raises its abdomen to draw a fibre between the droplet and the egg (Figure 4 EG).60 The fly stays in this position for 10 (~10% RH) to 35 seconds (~70% RH) to let the stalk dry and finally leaves.101 Introduction 16 Figure 4: Lacewing producing an egg stalk: A-C: The lacewing taps its abdomen several times on the substrate; D: A droplet of dope is deposited on the surface and the egg is pressed into the droplet; EG: The abdomen is pulled away from the substrate and a stalk is drawn. G: The fly holds its abdomen up until the stalk is hardened (approximately 10-35 seconds depending on the relative humidity); H: The stalk is finished. Introduction 17 3.1.4.2.4. The colleterial gland The origin of the lacewing egg stalk proteins is the colleterial gland of female lacewings which is located in the dorsal region of the sixth to eighth body segment and has a sac-like shape. The exit of the gland joins the fallopian tube (Figure 5).102 Figure 5: Schematic organisation of the female lacewings genitals. A: Colleterial gland; B: Fallopian tube; C: Receptaculum seminis; D: Paired ovary; E: Ovarioles; F: Egg. Lucas and Rudall showed by transmission electron microscopy that secretion of the colleterial gland of Chrysopa flava, which was diluted with water, comprised fibrillar structures. These structures did not solubilise during dilution.26 The fibrils are an assembly of around 20-30 molecules in side chain direction (about 13 nm) and have a thickness of approximately 2.5 nm which is the thickness of the fold of eight amino acids proposed by Geddes et al. and lengths of about 670 nm.26, 47 This preassembly in the spinning dope has been discussed to be a key requirement for a cross-ß structure in the stalk.26 Introduction 18 3.1.4.2.5. Protein sequences/dope composition Egg stalks of M. signata are composed of at least two proteins. The underlying c-DNA was extracted from a c-DNA library of colleterial gland cells.9 The two genes encoding the proteins (MalXB1 and MalXB2) are found to be expressed in a ratio of 7:1.9 Both proteins consist of a highly repetitive core domain with a repeat of 16 amino acids flanked by non-repetitive terminal domains. MalXB1 additionally has a non-repetitive central domain. MalXB1 is negatively charged with 17 acidic and 9 basic amino acid residues, whereas MalXB2 is positively charged with 11 acidic and 31 basic amino acids. An interesting fact is the presence of seven (MalXB1) and five (MalXB2) cysteine residues, which are mostly situated in the non-repetitive terminal domains. 3.1.4.2.6. Mechanics of lacewing egg stalks Mechanical tests on egg stalks were undertaken by Hepburn et al. (C. carnea) and Weisman et al. (M. signata).9, 103 Stress strain measurements at 65% relative humidity revealed extensibilities of ~249% and 381% and a tensile strength of ~375 MPa and 310 MPa. Measurements under water showed higher extensibility (502%/~560%) while strength was reduced (186 MPa/~250 MPa). Weisman et al. measured the lateral stiffness of egg stalks by scanning probe microscopy. Due to the lack of some physical constants of the system the modulus is reported in relation to B. mori silk. The measurements revealed 70% higher modulus values for the egg stalks. For the calculation of the bending stiffness, where the shape of a cross section is taken into account, the egg stalks have three times higher values (round shape of the stalks in comparison to the trilobal shape of B. mori silk).9 The high extensibility and high lateral stiffness might be related to the cross-ß structure and disulphide cross linking. Introduction 19 3.1.4.2.7. Structure The secondary structure of egg stalk proteins was investigated by Parker and Rudall.90 X-ray diffraction patterns showed cross-ß structure with ß-strands running perpendicular to the fibre axis, and were excitingly the first detected natural protein with this structure. 9, 90 1968 Geddes et al. proposed a structural model to explain the X-ray diffraction pattern.47 The stalk is built up by 25 Å thick micelles being separated by variable sized inter micelle spacing of 15 ± 4 Å (Figure 6). The longest dimension of the micelle is oriented parallel to the fibre axis. The ß-strands are predicted to have a length of eight amino acids whereof the first two and last two amino acids form a ßturn. In each turn one of the two central amino acids has to be a glycine.47 This prediction was confirmed by Weisman et al. for MalXB1 and MalXB2 where one glycine residue is found per turn in the repetitive domain. They further found that charged (Lysine) and bigger (>124 g/mol) amino acids are situated in the central turn regions.9 In MalXB2 two of the four central positions of the ß-strands are alanine residues. Figure 6: Cross-ß micelle of a lacewing egg stalk. Introduction 20 Recombinant production of silk proteins 3.2. In contrast to spiders it is possible to farm lacewings which are utilised on a commercial scale as a biological pest control.104 To obtain lacewing silk either flies have to be killed, the silk gland dissected, and the protein extracted, or single egg stalks have to be harvested, the eggs removed, the stalks solubilised, followed by extraction of the proteins. Both are time consuming and do not lead to large quantities of protein. Furthermore, both methods yield an undefined mixture of the proteins and other molecules such as the relatively low molecular weight egg defence molecules mentioned before, which are present in the stalk. Recombinant production of lacewing silk proteins in host organisms such as bacteria or yeasts is a possibility to obtain reasonable quantities of pure proteins with consistent quality, which is an essential requirement for the use of the proteins in industrial applications. An advantage is that single domains of the proteins can be produced, and their contribution to the assembly of the protein can be analysed. Another advantage of recombinant silk production is the possibility to alter the properties of the proteins. For example, the substitution of poly-alanine rich modules (crystalline leading to stiffness and strength) for coiled coil forming modules (more extensible) might lead to a more elastic fibre after spinning of the protein. Addition of signal peptides such as the integrin recognition motive RGD or cell penetrating peptides to silk proteins leads to new functions of the proteins such as improved cell adhesion.105-107 Commonly reported problems occurring during the recombinant production of silk proteins are low yields and truncated versions of the proteins. The reasons therefor are the size of silk proteins (protein yield decreases at protein sizes above 100 kDa in Escherichia coli (E. coli)),108 and their highly repetitive character (leads to undesirable recombination). Various attempts have been made to overcome these problems, for example optimisation of the gene sequence (less repetitive genes due to codon variation/adaption of the codon usage of the host organism), host engineering (changes in the metabolism of the host organism), and a modular approach leading to shortened versions of the proteins.108-115 Introduction 21 Many companies such as AMSilk GmbH (Germany), BASF AG (Germany), Commonwealth Scientific and Industrial Research Organization (Australia), and Spiber AB (Sweden) are interested in recombinant production of silk proteins for industrial applications. Technical processing of silk proteins 3.3. One particularly attractive feature of silk proteins is the possibility to produce different morphologies in addition to fibres. For other silk proteins, the production of fibres, particles, capsules, hydrogels, foams, films, and coatings has been reported, with possible applications ranging from drug delivery to high tech textiles. Allergan Inc. (USA), AMSilk GmbH (Germany), Commonwealth Scientific and Industrial Research Organization (Australia), Ekteino Laboratories Inc. (USA), Neurotex Ltd. (England), Orthox Ltd. (England), Oxford Biomaterials Ltd. (England), Spiber AB (Sweden), Spintec Engineering GmbH (Germany), Suturox Ltd. (England), and Vaxess Technologies Inc. (USA) work on processing silk proteins into marketable products such as conduits for nerve regeneration, coatings for breast implants, sutures, meniscal cartilage-, bone-, joint repair, and thermo stable vaccines. In this thesis some of the mentioned morphologies have been produced out of recombinant lacewing silk, and are therefore introduced briefly. Fibres For the processing of silk proteins (recombinant as well as regenerated natural silk) into fibres there are a few techniques such as hand drawing, wet spinning,116-125 microfluidic spinning,92, 126 or electro spinning.127-131 In this thesis the fibres were hand drawn. Hand drawing fibres requires a highly concentrated protein solution which can be either aqueous or non-aqueous (Hexafluoroisopropanol (HFIP) or Hexafluoroacetone trihydrate (HFA) have been used). To produce a fibre, a droplet of silk solution is deposited on a surface and a fibre is pulled out of the droplet by using tweezers, and subsequently Overview of the thesis including unpublished data 28 After rearrangement of the strands at 100% relative humidity, new hydrogen bonds can be formed. This results in an extension of the stalk of up to 500%. Finally the ß-strands start slipping on each other by a stick-slip mechanism described by Keten et al. before the stalk ruptures.187 In contrast at 30% relative humidity the hydrogen bonds have higher bond energy due to the lack of interacting water molecules. Therefore, in one layer of the stalk the sum of hydrogen bonds has higher bond energy than the individual disulphide bonds which crosslink the proteins in the stalk. The disulphide bonds break and the stalk ruptures without breakage of the hydrogen bonds along the entire length of the stalk. Silk gland analysis of lacewings 4.3. Colleterial glands of female C. carnea were dissected under a 95 mM sodium chloride (NaCl) solution and incubated three times in fixing buffer (100 mM HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid), 2.5% glutaraldehyde, 2% formaldehyde, pH 7.2) (one hour, two hours, 18 hours in the fridge respectively). Afterwards the glands were washed three times with wash buffer (100 mM HEPES, pH 7.2) followed by incubation in 1% osmium tetroxide (v/w in water) for one hour at 4 °C and three times washing in water. After embedding the glands in 1% agar they were dehydrated using increasing concentrations of ethanol (30%, 50%, 70%, 90%, and two times 100%). Ethanol was substituted by incubating the embedded gland in a 1:1 mixture of ethanol and propylene oxide (15 minutes), followed by incubation two times in pure propylene oxide (12 hours at -20°C). Finally the samples were embedded in glycid ether 100 using standard procedures. The samples were cut with a diamond knife equipped ultramicrotome into ultrathin sections, which were mounted on copper grids. After staining with uranyl acetate and lead citrate they were imaged using a transmission electron microscope (TEM). Two to four cell layers were detected depending on the position in the gland. In general the gland cells contain many mitochondria and are filled with rough endoplasmic reticulum (Figure 9). In some cells vesicles with fibrillar structures are visible (Figure 9 D-F). The fibrils have diameters of about 5 nm and lengths of about 200 nm. This may be related to egg stalk proteins forming eight amino acid ß-strands. Such a structure would have a thickness of 2.5 nm. Lucas and Rudall showed similar structures in diluted dope from a Overview of the thesis including unpublished data 29 Chrysopa flava colleterial gland.26 Such a pre-structured silk solution might be critical to achieve a cross-ß structure. The fibrils might be oriented by shear forces during the egg stalk production, and the cysteines might cross-link the fibrils to give a stable egg stalk structure (Figure 15). Figure 9: TEM images of cross sections of a lacewing colleterial gland; A: Silk gland tissue and silk dope (arrows); B: Cells with Nucleus (n), mitochondria (m), and silk dope; C: Inhomogeneity of the silk solution; D, E and F: Cells are filled with rough endoplasmic reticulum (rer) and protein filled vesicles with fibrillar structures (f). Overview of the thesis including unpublished data 30 Figure 10: Silver stained SDS-page of silk solution stored in the colleterial gland of a female lacewing (C. carnea). M: protein marker; G: silk gland extract. SDS-PAGE analysis of the silk dope of a female C. carnea revealed five distinct bands (Figure 10). Surprisingly this are three more than Weismann et al. gathered from their c-DNA library of M. signata.9 One reason for this might be the quality of their c-DNA library where only one clone encoding MalXB2 and seven clones encoding MalXB1 could be found. Biotechnological production of N[AS]8C, an 4.4. artificial lacewing egg stalk protein Due to the low availability of lacewing egg stalk protein from natural sources, it is an important step to produce the proteins in a different manner, for use in both basic research and technical processing. Here we developed an artificial version of MalXB2, an egg stalk protein of M. signata and produced it recombinantly in E. coli bacteria. MalXB2 consists of non-repetitive aminoand carboxy-terminal domains. These were used as modules N (amino-terminal module) and C (carboxy-terminal module) for the artificial Protein N[AS]8C (Figure 11). To ensure a proper steric build-up of the protein (the terminal domains in the natural blueprint face to different sides of the cross-ß sheets of the repetitive part (Figure 6)) the carboxy-terminal module starts with the last eight amino Overview of the thesis including unpublished data 31 acids of the repetitive part of MalXB2 (Figure 11). Detailed analysis of the amino acid sequence of the repetitive part of MalXB2 showed a repeat every 48 amino acids, which was called AS module and better fits to the original sequence than a 16 amino acid repeat proposed by Weismann et al.. 9 The starting of the 48 amino acid repeat was chosen to start with a glycine which is relevant for the use of a cloning strategy developed by Hümmerich.109 The modules were translated to E. coli optimised nucleotide sequences and an additional nucleotide triplet (GGC) was added to the end of the Nand AS module (Due to the cloning technique. These nucleotides get lost during multimerisation/cloning). The cloning technique allows a seamless multimerisation of the AS modules and subsequent linkage of the Nand C module (Figure 11 C). As a length of the repetitive part we chose eight AS modules to mimic the original length. Afterwards the N[AS]8C nucleotide sequence was cloned into a pET 28a vector. E.coli BL21 (DE3) cells were transformed with the plasmid and were cultivated in a 2.5 L fermenter. After induction and expression of the artificial gene, the bacteria could be harvested and a purification strategy for the protein had to be developed. The bacteria were lysed by an urea/thiourea buffer followed by pH decrease to 4. Finally the protein was purified without the use of columns by fractionated ammonium sulphate precipitation, and after washing with water, lyophilised and stored at -20°C. Purity was tested by SDS-PAGE and mass spectrometry (Figure 12). Overview of the thesis including unpublished data 32 Figure 11: A: Sequence and organisation of MalXB2, a natural egg stalk protein; B: The three modules derived from MalXB2; C: Schematic build-up of an artificial lacewing egg stalk protein - N[AS]8C. The full length protein N[AS]8C has a molecular weight of 53 kDa including detection and purifications tags and is comparable in size to the naturally occurring variant MalXB2 (55 kDa). Overview of the thesis including unpublished data 33 Figure 12: SDS-PAGE (A) and mass spectrometry (B) of N[AS]8C. The lower molecular weight peaks are multiple charged N[AS]8C molecules. Adapted with permission from Angewandte Chemie International edition (2012, 51, 6521-4). Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Fibre/stalk formation and analysis 4.5. To produce fibres out of N[AS]8C, we used a process close to the natural one. Due to the low solubility of the protein in water we chose HFA as the solvent. The volatility of HFA is beneficial for fast drying of the generated fibres. 10% w/v of lyophilised N[AS]8C was dissolved in HFA to produce a solution from which fibres could be spun. A small droplet (~1-2 µL) was pipetted on a surface. Now tweezers were dipped into the droplet, mimicking the lacewing egg stalk production, and were subsequently pulled out to draw a fibre. The end connected to the tweezers was transferred to a tinfoil support (Figure 13 A). After drying, the tinfoil support was moved closer to the droplet to reduce tension in the fibres and avoid rupture during post treatment. By applying 60°C and 70% relative humidity overnight the ß-sheet content increased from 20% to 32% which is close to the values of natural egg stalks with 40% ßsheet content and the fibres turned water insoluble. Overview of the thesis including unpublished data 34 Figure 13: Comparison of natural and artificial stalks. A) schematic picture of the stalk production 1) stalk drawn from the dope to tinfoil using tweezers; 2) relaxation of the stalk by moving of the tinfoil; 3) the stalk contracts during post-treatment at 60°C and 70% relative humidity; B) picture of an artificial egg stalk with tinfoil on top (left) and a natural egg stalk with an egg on top (right). Reprinted with permission from Angewandte Chemie International edition (2012, 51, 6521-4). Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. The artificial stalks are stiff, like the natural ones (Figure 13 B). Analysis of the artificial stalks showed similar mechanical properties to the natural ones at 30% relative humidity with 2% extensibility for the natural ones compared to 5% for the artificial ones (Table 3). Even the tensile strength is nearly equal with 55 MPa compared to 68 MPa for the natural stalks. Nevertheless there are differences at 70% relative humidity where the extensibility of the natural stalks of 210% could not be observed for the artificial ones. Artificial stalks show, as well as the natural lacewing egg stalks, birefringence under a polarised microscope, meaning there are ordered structures in the stalk (Figure 14). Nevertheless, no cross-ß structure could be detected by X-ray diffraction or by polarised FTIR (Fourier transform infrared spectroscopy) measurements. This might be a reason for the differences in mechanical properties at 70% relative humidity. In the absence of crossß structure no transition to parallel-ß structure is possible. This means the stalks will not be that extensible and the mechanical properties will differ. Overview of the thesis including unpublished data 35 Table 3: Tensile testing of natural (C. carnea) and artificial egg stalks. Experiments were carried out at 30% and 70% relative humidity at 22°C. Reprinted with permission from Angewandte Chemie International edition (2012, 51, 6521-4). Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Figure 14: Comparison of birefringence of natural and artificial stalks. A) Microscopic pictures of a natural lacewing egg stalk. B) Microscopic pictures of an artificial egg stalk. Top: bright field; bottom: with crossed polarisers; scale bars: 50 µm. Reprinted with permission from Angewandte Chemie International edition (2012, 51, 6521-4). Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Extensibility [%] Strength σmax [MPa] Young’s modulus [MPa] Toughness [MJ/m³] Literature values (M. signata and C. spec) 65% RH9 381 310 65% RH103 249 ~375 Natural egg stalk 30% RH 2 ± 1 68 ± 19 5,777 ± 1,257 1.2 ± 0.72 70% RH 210 ± 100 155 ± 75 3,175 ± 1,016 87 ± 49 Artificial egg stalk 30% RH 5 ± 2 55 ± 14 2,330 ± 850 1.76 ± 0.9 70% RH 6 ± 3 25 ± 11 1,012 ± 252 1.09 ± 0.59 Overview of the thesis including unpublished data 36 A possibility to solve this problem in the future is to use an aqueous spinning solution with pre-structured proteins, as seen for natural dope (Figure 9), which might orient due to shear forces during the production process and crosslink to each other by disulphide bonds (Figure 15).26 Figure 15: Model of a pre-structured silk solution which assembles upon shear forces. Overview of the thesis including unpublished data 37 Further processing of a recombinant lacewing 4.6. protein Next the processing of N[AS]8C into different morphologies was tested as described for other silk proteins. 4.6.1. Films In order to obtain films (Figure 16) 1% (w/v) N[AS]8C was dissolved in HFA or formic acid. Both solutions were cast on polystyrene. After drying, the films were either peeled off the substrate or first post-treated by immersion in methanol or over-night treatment at 60°C and 50% relative humidity. Secondary structure was analysed using FTIR. For SEM the samples were glued on aluminium stubs and were sputter-coated with platinum. Images were obtained using a Zeiss 1530. Table 4: Secondary structure of films cast from HFA and formic acid. The HFA films were post-treated with methanol or at 60°C and 50% RH overnight. HFA Formic acid As cast Untreated after 4 days 60°C 50% RH over night MeOH treated As cast ß sheets 6% 19% 42% 50% 59% Alpha-helices 21% 12% 9% 7% 6% Turns 11% 26% 18% 22% 14% Random coils 58% 39% 23% 20% 20% Side chains/ aggregated strands 4% 4% 8% 1% 1% Films cast from formic acid (Figure 16 B, D, F) were more brittle than the ones from HFA, and water insoluble directly after drying. They had a ß-sheet content of approximately 59% (Table 4). In contrast, films cast from HFA (Figure 16 A, C, E) were water soluble and, therefore, had to be post-treated. Without post-treatment they had a ß-sheet content of 6% which increased due to aging to around 19% (after 4 days). Overnight heat treatment of the films at 60°C and 50% relative humidity led to a ß-sheet content of around 42%. Slightly higher values could be obtained by treating the film with Overview of the thesis including unpublished data 44 non-adherent. 94% of the cells grew on the ntagCysC16-c(RGDfK) stripes (Table 5 and Figure 23). Surprisingly fibroblasts grown on structured control films of unmodified eADF4(C16) with N[AS]8C as a top layer and structured film only out of eADF4(C16) showed the same tendency of distribution with 92% respectively 85% of the fibroblasts in the grooves (Table 5 and Figure 23) but with much lower overall cell number on the solely eADF4(C16) films. Figure 23: BALB/3T3 fibroblasts grown on structured films. A: Orientation of fibroblasts grown on patterned films made of different protein combinations (ground layer protein/ ridge protein) as depicted by the colour code after 48 hours of incubation; B: Fluorescence microscopy of calcein AM (Calcein acetoxymethyl ester) stained cells, grown on a film with ntagCysC16-c(RGDfK) as ground layer and N[AS]8C as ridges; C and D: light microscopic image after 48 hours of incubation using ntagCysC16-c(RGDfK) as ground layer with N[AS]8C as ridges (C) and eADF4(C16) as ground layer with N[AS]8C as ridges (D). Adapted by permission of The Royal Society of Chemistry. Overview of the thesis including unpublished data 45 The orientation of the fibroblasts was most pronounced for films out of eADF4(C16) with 80.6% of the cells being oriented in an angle of ±7.5° to the structures axis whereas only 52.1% of the fibroblasts on ntagCysC16-c(RGDfK)/N[AS]8C were in this range (Figure 23 A). This indicates the importance of the structure to orientate the cells while the materials/proteins properties influenced their location. Even after 96 hours of cultivation the fibroblasts stayed mostly on the ntagCysC16c(RGDfK) protein stripes and proliferated well to a high cell density (Figure 24 A). Such high cell densities are necessary for many tissue culture experiments, such as differenciation of myoblasts into myotubes. C2C12 myoblasts showed the same tendency as the fibroblasts, but we were not able to count cells and measure their orientation to the scaffold due to their ability to form myotubes and higher cell density (Figure 24 B). Figure 24: A: BALB/3T3 fibroblasts grown on structured films using eADF4(C16) as ground layer and N[AS]8C as ridges after 96 hours of incubation; B: C2C12 myoblasts grown on eADF4(C16)/N[AS]8C films after 48 hours of incubation. Reproduced by permission of The Royal Society of Chemistry. Such films might be used in/as scaffolds for tissue engineering of tissues, where an ordered structure of the cells and high selective cell density is of advantage. Skeletal muscles as well as bone or epithelial cultures might be possible applications. Overview of the thesis including unpublished data 46 Individual contributions to joint publications 4.8. Chapter 7 Chapter 6 is reprinted with permission from Biomacromolecules (2012, 13, 3730-5). Copyright 2012 American Chemical Society. “Dependence of Mechanical Properties of Lacewing Egg Stalks on Relative Humidity” By Felix Bauer, Luca Bertinetti, Admir Masic, and Thomas Scheibel I carried out all of the measurements except the RAMAN measurements. Luca Bertinetti and Admir Masic performed the RAMAN measurements and corrected the manuscript. Felix Bauer and Thomas Scheibel wrote the manuscript. Chapter 8 Chapter 7 is reproduced with permission from Angewandte Chemie (2012, 51, 6521-4). Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. “Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein“ By Felix Bauer and Thomas Scheibel I carried out all the design, cloning, fermentation, purification and measurements. Felix Bauer and Thomas Scheibel wrote the manuscript. Chapter 9 Chapter 8 is reproduced by permission of The Royal Society of Chemistry. This work is submitted for publication: Biomaterials Science (2013, 1,1244-9). “Controllable cell adhesion, growth and orientation on layered silk protein films” By Felix Bauer, Stefanie Wohlrab, and Thomas Scheibel. I developed the technique to fabricate the striped protein films, and carried out the image analysis after cell culture. Stefanie Wohlrab carried out the cell culture experiments. Felix Bauer, Stefanie Wohlrab and Thomas Scheibel wrote the manuscript. Literature 47 5. Literature 1. Sutherland, T. D.; Young, J. H.; Weisman, S.; Hayashi, C. Y.; Merritt, D. J., Insect silk: One name, many materials. 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Arbitary unit B. mori Bombyx mori C. carnea Chrysopa carnea Da Dalton E. coli Escherichia coli FTIR Fourier transform infrared spectroscopy HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HFA Hexafluoroacetone trihydrate HFIP Hexafluoroisopropanol M. signata Mallada signata MeOH Methanol PDMS Polydimethylsiloxane RGD Arginine-glycine-aspartic acid RH Relative humidity SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Scanning electron microscopy TEM Transmission electron microscope Tris 2-Amino-2-(hydroxymethyl)-propan-1,3-diol w/v Mass/volume Dependence of Mechanical Properties of Lacewing Egg Stalks on Relative Humidity 65 7. Dependence of Mechanical Properties of Lacewing Egg Stalks on Relative Humidity Dependence of Mechanical Properties of Lacewing Egg Stalks on Relative Humidity 66 Dependence of Mechanical Properties of Lacewing Egg Stalks on Relative Humidity 67 Dependence of Mechanical Properties of Lacewing Egg Stalks on Relative Humidity 68 Dependence of Mechanical Properties of Lacewing Egg Stalks on Relative Humidity 69 Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein 76 Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein 77 Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein 78 Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein 79 Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein 80 Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein 81 Artificial Egg Stalks Made of a Recombinantly Produced Silk Protein 82 Controllable cell adhesion, growth and orientation on layered silk protein films 83 9. Controllable cell adhesion, growth and orientation on layered silk protein films Controllable cell adhesion, growth and orientation on layered silk protein films 84 Controllable cell adhesion, growth and orientation on layered silk protein films 85 Acknowledgement 93 11. Acknowledgement Danksagung Diese Arbeit wurde von Juli 2008 bis Mai 2013 am Lehrstuhl Biomaterialien der Universität Bayreuth unter Betreuung von Prof. Dr. Thomas Scheibel durchgeführt. Ihm möchte ich ganz herzlich für die Möglichkeit danken, dieses spannende Thema mit vielen Freiheiten und Möglichkeiten zu bearbeiten. Prof. Dr. Thomas Scheibel, Prof. Dr. –Ing. Volker Altstätt und Dr. Stefan Geimer danke ich für die Übernahme meines Mentorats. Für die Einweisung und Hilfen sowie Aufnahmen am REM und TEM danke ich Dr. Rainer Detsch, Johannes Diehl, Claudia Blüm, sowie Dr. Stefan Geimer und Kristin Schacht. Admir Masic und Luca Bertinetti danke ich für die Zusammenarbeit bei der Publikation und die Messungen am Raman. Stefanie Wohlrab will ich für die gute Kooperation bei der Arbeit für unsere Veröffentlichung danken. Prof. Louise Serpell und Kyle Morris gilt mein Dank für ihre Bemühungen mit der Aufnahme von Röntgenbeugungsspektren, auch wenn diese nicht den Weg in diese Dissertation gefunden haben. Ute Kuhn danke ich für die TGA und DSC Messungen. Annemarie Heiduk danke ich für die Unterstützung bei den Zugmessungen an Köcherfliegenseide im Rahmen ihrer Projektarbeit. Natürlich möchte ich mich auch bei meinen Kollegen bedanken: - Susanne Schramm für ihre immer freundliche und aufmunternde Art. - John für die Hilfestellungen in der Anfangszeit und die lustigen Abende nach dem KondiTraining mit isotonischem Sportgetränk. - Lukas, Martin, Andrew für das Korrekturlesen der Paper und Diskussionen und Tipps wenn mal Probleme auftraten. - Klonmaster Anderl für die Ausbildung als sein Padawan. - Anderl und Johnobo für ihre Hilfe und Unterstützung beim Fermentieren. - Den Stammkunden im BBB. Acknowledgement 94 - Den Fußballern für die lustige Ablenkung auch in stressigen Zeiten. - Den Kellerkindern. - Bei meinen Bürokollegen David, Gregor, Kristina, Anja, Markus, Aniela, Elena und Claudia dafür, dass sie mich und meine Launen ertragen haben und wir immer eine lustige Zeit miteinander hatten. Zu guter Letzt möchte ich meinen Eltern danken, dass sie mich über all die Zeit unterstützt haben und mit immer den Rücken frei gehalten haben. Erklärung 95 12. Erklärung: Hiermit erkläre ich eidesstattlich, dass ich die vorliegende Arbeit selbständig verfasst, und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe. Ferner erkläre ich, dass ich weder an der Universität Bayreuth noch anderweitig mit oder ohne Erfolg versucht habe, eine Dissertation einzureichen oder eine Doktorprüfung abzulegen. Des Weiteren erkläre ich, dass ich weder bisher noch in Zukunft Hilfe von gewerblichen Promotionsberatern bzw. –vermittlern in Anspruch genommen habe bzw. nehmen werde. Bayreuth im Mai 2013 …………………………. Felix Bauer