| Orb-weaving spider utilize up to six different protein-based silks, each secreted from a specialized gland, and each evolved to fulfill a certain task. The minor ampullate silk, used for auxiliary spiral and prey wrapping, displays intriguing mechanical properties and excellent biocompatibility. Particularly, minor ampullate silk does not supercontract when hydrated, which thereby avoid an obvious drawback if implantation is aimed for. Minor ampullate silk is therefore considered to be a strategic resource that can be developed for applications in high-technology fields including military, aerospace and biomedical areas.Due to the cannibalism of most spiders and small amounts of silks, large-scale production of spider silk is not possible by housing spiders at high densities, as for silkworms, which limits their utilization. Since1990scientists have tried to move the application of spider silks forward by biotechnological means, in order to exploit the outstanding fibers in modern technology. However, biomimetic spider silk with similar properties to native silk has not yet been developed, delaying the industrialization of this novel strategic resource.Experience from the previous work shows mainly three bottlenecks in producing native-like spider silks artificially:1) Full-length gene cloning of spider silk proteins (spidroins) is limited. Genes coding for spidroins are extraordinarily large and repetitive with high GC content, and complete gene characterization by PCR and cDNA library is not practically feasible. From1990to2014, only three spidroin full-length genes have been sequenced (the second one is from this dissertation);2) Recombinant production of complete spidroins is not yet achieved. Spidroins are up to300kDa in size and pronouncedly modular (repetitive) with high GC content in the coding genes, which generally cause problems such as genetic instability and unwanted mRNA secondary structures. Also, the high frequency of Gly and Ala makes the spidroins difficult to express in heterologous hosts;3) Silk formation mechanism is still not unraveled. The extremely small inner diameter (micron-sized) complicates the measurements of the physiological and chemical microenvironments of silk secreting glands. Furthermore, spidroins are highly repetitive in sequence but capped by non-repetitive N-and C-terminal domains (NT and CT), and it is to date not known in detail what roles the terminal domains play in the silk formation process. Additionally, the structure and function of the unique spacer domains in minor ampullate spidroin (MiSp) are entirely unknown. Compared to the microenvironments. of silk glands and native silk formation mechanisms, the spinning technologies used so far are primitive.In order to remove the three bottlenecks above for generating high-performance biomimetic spider silks, in this dissertation Araneus ventricosus MiSp was studied firstly in order to lay the foundations for preparation of artificial spider silks with superior properties:Part one:full-length MiSp gene characterization. A. ventricosus, one of the spider species widely distributed in China, the silks of which exhibit striking performance, has been used for spidroin gene characterization. Only partial cDNA sequences for Minor ampullate silk have so far have been obtained, and recombinant expression and biomimetic silk preparation have not even been attempted. In order to enable recombinant production of MiSp and native-like minor ampullate silk spining, and also provide a new resource for research on spidroin evolution and gene regulation, I have characterized the A. ventricosus MiSp full-length gene. Based on the previous A. ventricosus fosmid gene library and STS/3D-PCR screening method, we obtained a positive clone containing the full-length A. ventricosus MiSp gene. The insert, about33kb, encompasses the full-length MiSp coding sequence as well as6647bp upstream of its start codon and14937bp downstream of its stop codon. The complete MiSp gene is composed of two exons and one unusually large intron, which is a novel finding for spidroin genes. The single intron in A. ventricosus MiSp DNA is5628bp in size and begins with the nucleotides "GT"(guanine, thymine) and ends with "AG"(adenine, guanine) and thus follows the GT-AG rule. The spliced full-length transcript of A. ventricosus MiSp coding gene is5440bp in size and encodes1766amino acid residues organized into conserved nonrepetitive N-and C-terminal domains and a central predominantly modular region (more than90%). A. ventricosus MiSp repetitive region (modular region), mainly consist of four motifs (Gly-X, Gly-Gly-X, Gly-Gly-Gly-X and poly-Ala) that are iterated in a non regular manner, and are interrupted by two nonrepetitive spacer regions (126residues), which share100%identity even at DNA level. Identification of the first full-length MiSp gene sequence reveals an unusually variable repetitive part, extremely conserved spacer regions, that the exon-intron organization differs between all so far characterized spidroin genes, and finally a new conserved element CACG for transcript regulation. Being the first full-length MiSp sequence (and the second full-length spidroin sequence), A. ventricosus MiSp full-length sequence fills a gap and provides a new gene blueprint for full-length MiSp recombinant production and biomimetic silk formation.Part two:Structure and function of MiSp NT and CT, and silk assembly mechanism. Spider silk fibers are produced from soluble concentrated spidroins under ambient conditions in silk glands in a poorly understood complex process, in which physiological, physical and chemical conditions change progressively. Spidroins are highly repetitive in sequence but capped by non-repetitive NT (～130aa) and CT (～110aa) domains that have been suggested to regulate silk formation in similar manners (a hypothetic model from major ampullate spidroin, MaSp). In this dissertation, I show that the terminal domains from A. ventricosus MiSp respond in opposite ways to pH changes. For each spidroin, mainly the Rp region determines the mechanical properties, while NT and CT domains play very important roles in regulating the spidroin to silk transformation. Besides the three typical domains, A. ventricosus MiSp sequence contains two spacer domains of unknown function. From our recent work, the pH gradient in Nephila clavipes major ampullate gland has been determined and is found to be much broader than previously known, and the concentrations of some irons were also measured. Interestingly, carbonic anhydrase, which catalyses CO2+H2O←'HCO3-+H+, was unexpectedly found in the gland and it maintains the pH gradient. Herein, comprehensive studies of A. ventricosus MiSp NT, CT and spacer domains showed:1) MiSp NT dimerizes progressively when pH decreases form7.5to6.0, and the subsequent stabilization of NT dimers between pH6and5will result in the firm locking of spidroins into multimers in the distal part of the duct. NaCl in a physiological concentration shifts the dimerization process to lower pH. A. ventricosus MiSp NT is composed of5a-helices, Glu115makes an intramolecular salt bridge with Arg64, and cysteines located in helix1and4form an intramolecular disulfide bond. Glu84of Euprosthenops australis MaSp1NT, which is involved in the last step of dimer stabilization, lacks a counterpart in A, ventricosus MiSp NT. However, Asp109in MiSp NT is conserved, which possibly indicates a novel mechanism for MiSp NT dimerization;2) MiSp CT adopts a dimer conformation and, in sharp contrast to MiSp NT, it is gradually destabilized when pH is lowered from7.5to5.0. At pH5.5and below, CT converts to a thioflavin T (ThT) positive state, which was not observed at higher pH, or for NT at any pH tested, and NaCl delays CT (3-sheet amyloid fibril formation. Characterization of the ThT positive aggregates by TEM and Congo red staining showed typical amyloid-like fibrils, which can trigger rapid polymerization of the spidroins. Low pH and increasing HCO3-concentration implies that pCO2is elevated along duct. The CO2analogue CS2interacts with specific, mainly nonpolar, residues distributed in helices2-4of A. ventricosus MiSp CT. Similar to MiSp NT, MiSp CT monomer is also composed of5a-helices, dimerizes mainly by hydrophobic interactions, and Arg43makes an intramolecular salt bridge with Glu87. Carbonic anhydrase activity, and NT and CT opposite stability changes emerge in the same region of the gland, suggesting a novel CO2dependent trigger mechanism of spider silk formation;3) In contrast to MiSp NT and CT domains, A. ventricosus MiSp spacer domain, folds into an a-helical conformation under different pH, and shows the same Tm of～50℃between pH7and5. The main quaternary structure of A. ventricosus MiSp spacer is a tetramer (small amounts of monomers are observed), which may be a way to connect spider silk protein molecules. NaCl can promote the spacer monomer, and prohibits tetramer formation. Interestingly, MiSp spacer self-assembles into short fibers visible to the naked eye, indicating that A. ventricosus MiSp spacer domain is able to form silks and may promote formation of solid silk fibers. According to the detailed determination of A. ventrisocus MiSp NT, CT and spacer domains, a novel spider silk formation mechanism has been suggested, which may lead to novel technology for biomimetic high-performance spider silk generation.Part three:Directional ligation of full-length MiSp. Complete spidroin is one of the decisive factors for high-performance artificial spider silk preparation, however, so far recombinant spider silk proteins are based on partial genes or a few tandem repetitive motifs. As expected, biomimetic fibers made from such spidroins are inferior to native spider silk as regards mechanical properties. Different kinds of expression hosts have been tried in recent years, but no one has been found to support the expression of complete spidroin genes. A. ventricosus MiSp coding gene is extremely large, and Gly/Ala accounts for a huge proportion, making it very difficult to express complete spidroin in Escherichia coli. Here Rosetta2(DE3) was used firstly as host to try to express A. ventricosus MiSp full-length gene, but on SDS-PAGE no full-length protein was found, but lots of truncated MiSp proteins. In order to find an efficient method for preparing complete spider silk proteins, full-length A. ventricosus MiSp proteins were firstly directionally ligated, mediated by Rb intein. Full-length MiSp coding gene was divided into two fragments, and fused with Rbn and Rbc respectively which can be expressed in E. coli. The obtained fusion proteins are AvMiSpNTRbn and RbcAvMiSpCT. AvMiSpNTRbn and RbcAvMiSpCT were mixed at room temperature at equal proportions, whereby Rbn and Rbc should recognize each other in the presence of low concentration of DTT, and undergo a trans-splicing reaction (takes several hours), in which AvMiSpNT and AvMiSpCT are ligated in vitro into full-length MiSp via a peptide bond. The directional ligation of full-length A. ventricosus MiSp recombinant proteins mediated by intein is a novel in vitro technology, and make it possible to generate complete (high molecular weight). spider silk proteins, which is very useful for generating high-performance native-like spider silk preparation.In this dissertation, the first-ever comprehensive and systematic characterization of A. ventricosus MiSp was carried, in order to remove bottlenecks in spider silk biomimetic preparation. The first full-length MiSp coding gene identification, a novel spider silk formation mechanism dependent on CO2, and a novel directional in vitro ligation strategy for generation of complete spider silk proteins are presented. These results lay solid foundations for novel biomimetic MiSp fiber generation, and also for production of other spidroin-based fibers. |
PDF Full Text Request