| Humans have used spider silk as a material long before it became the focus of scientific research studies. In ancient Greece, natural cobwebs were used to seal bleeding wounds, and later, spider silks were utilized for military purposes, in particular in constructing crosshairs for gun sights. Recent studies have found that spider silks are very promising biomaterials with many potential uses in medical and other fields, such as tissue engineering, drug delivery and nerve conduit, because of their exceptionally high tensile strength, elasticity, toughness and light weight. Their properties surpasses all manmade and nature fibers, including silkworm silk.Despite significant interest in spider silk, silkworms have been the most frequently used source of protein silk. One reason that spider silk has lagged behind is that silkworms are fairly easy to domesticate, whereas spiders cannot be housed in high densities because of their carnivorous nature. In addition, whereas one silkworm cocoon yields600to900m of fiber, only～137m of fiber can be reeled from the ampullate gland of a spider and only～12m of silk is found in a complete spider web. Therefore, the best option for moving the application of spider silks forward is the developing of biotechnological means of artificially generating source material. But there are several challenges in expressing engineered silk protein in host cells because spidroins are composed of highly repetitive sequences that are rich in glycine and alanine and the prevalence of these codons in the mRNAs, appears to have caused the coevolution of tRNA pools in the spider host specially designed to deal with Gly-Ala-rich messages. In addition, the high GC nature of the DNA causes secondary-structure problems that lead to rearrangement and deletion artifacts when attempting to cloned cDNAs and genes. So the protein yield is usually very low and the spidroins produced are much smaller than natural forms and consequently the properties are not comparable to native spider silks. Another reason is that too little is known about spidroin protein composition as well as the assembly mechanism, because of its complexity, and so spinning using reconstituted silk results in poor fiber properties because of the non-natural way of spinning. In recent years, people have tried to solve these problems and have made significant advances. Studies on spider silks can be divided into five categories:(1) Identifying gene sequences of spider silk proteins.(2) Production of recombinant spidroins in heterogeneous cells.(3) Studying the mechanism of silk formation.(4) Examining the relationship between structure and function.(5) Improving the properties of recombinant silk fibers.Female orb-weaving spiders can produce up to six different silks, and as well a silk-like glue, in specialized glands with specific tasks, such as for constructing silk webs, wrapping preys and making egg cases. To date, most research has focused on the major ampullate gland, which manufactures dragline silk. Spiders use dragline silk to create web anchors, as well as for safety-lines for survival. It is extremely tough and has been revealed to have a tensile strength that is comparable to Kevlar (4×109N/m2), coupled with a reasonably high viscoelasticity (dragline35%, Kevlar5%). The minor ampullate gland, which shares morphological similarity to the major ampullate, synthesizes web radii filaments and temporary capture silk. Flagelliform gland silk is extremely extensible and forms the capture spiral of an orb web. It can extend to three times as long as its original length without breaking. Spider wrapping silk, also known as the aciniform silk, has the highest toughness among spider silks and is renowned for its ability to absorb energy without failing, due to a combination of high tensile strength and high elasticity (extensibility). For example, Argiope trifasciata wrapping silk is renowned for its ability to absorb energy without failing, being50%tougher than the tough dragline silk. In this thesis, I present the first studies on recombinant wrapping silk proteins.Identifying the DNA sequences encoding spider silk proteins is essential for producing recombinant spider silk. In this thesis, a coding sequence of one single repeat of wrapping silk was made as a synthetic gene based on the consensus repeat sequence of Argiope trifasciata AcSp1. In addition, its coding sequence was designed for optimal codon usage in E. coli without altering amino acid sequence. The coding sequence was flanked by specific restriction enzyme sites which allowed seamless ligation of DNA segments so that different numbers of repeats could be constructed. In addition, an H6-SUMO tag was added to the N-terminus of the silk protein construct to permit affinity purification and subsequent tag removal and to increase protein expression and solubility. This strategy yielded approximately10-40mg of the fusion proteins from each liter of the E. coli cell culture, depending on the protein sizes.Several eukaryotic systems, including yeast, plants, and cultured insect or mammalian cells, have been used to express recombinant spider silk proteins. However, these eukaryotic expressions attempts were plagued by small protein size, low yield, low solubility and/or high cost. Escherichia coli (E. coli) is a preferred, easy and low-cost, host cell for spidroin production, but it usually can produce only relatively high yields for small fragments of spider silk proteins. Only one example of successfully expressing native-sized dragline silk in metabolically engineered Escherichia coli has been reported. However, the expression level was relatively low. So it is very useful to study other methods to produce native-sized spidroins. In this thesis I constructed and applied a new strategy to produce native-sized wrapping silk by employing intein splicing. It is a new method of making large silk proteins and may be applicable to other silk proteins as well.There are two proposed models for silk formation. The liquid-crystalline theory proposes that rod-shaped spider silk proteins first adopt a nematic liquid-crystalline phase within the dope, The conformational transition of the silk proteins from random-coil and helix conformations to mainly p-sheet-rich structures is promoted by elongational flow and shear forces. The micelle theory proposes that spidroins form micelles in solution and these micelles coalesce to form larger globular structures. The force-field created by elongational flow and ductal wall boundaries elongates the globular structures, shaping them into fibrillar morphologies. These fibrillar structures are thought to be the precursors of the subsequent spider silk fibers. Most of the studies on spider silk protein, including of silk forming mechanisms have focused on major ampullate (MA) silk. It has been demonstrated that during the MA silk forming process, there is an increase in potassium and phosphate concentration and decrease in sodium and chloride concentration in the silk gland. Whether those chemical environment changes are universal, so for other types of silk, is still unknown. In this thesis, I studied recombinant wrapping silk protein (AcSpl) structure and assembly, by scanning electron microscopy, transmission electron microscopy, atomic force microscopy, circular dichroism spectrometry, raman spectrometry, nuclear magnetic resonance and with a nano-material properties tester. I found the minimal repeat unit for fiber formation of wrapping silk to be four repeats and measured its average strength (115MPa), which is1/6that of native wrapping silk, but higher than previous reported for recombinant dragline silk. The recombinant proteins were found to form micelles in solution and align to form fibrils under shear forces. Micro-fibrils assemble to form larger macro-fibrils and finally form fibers after dehydration. The process we observed supports the micelle theory of fiber assembly. There is also a structure transition after the fiber is formed, from predominantly a-helical in solution to P-sheet in the fiber, which is very similar to the situation in native spider silk fiber.Most spidroins are composed of a large core repetitive domain, a N-and C-terminal nonrepetitive domain. The C-terminal nonrepetitive domain is more conserved compared to the core repetitive domain among different spidroins. It is proposed to be essential for fiber formation and organized orientation of silk protein molecules. We found the C-terminal domain helped recombinant AcSp1fiber formation and improved fiber mechanical properties.This research has produced a new method for studying self-assembly mechanisms of spider silk proteins in forming silk fibers and structure and function relationship and provided a unique opportunity for comparing two different classes (aciniform vs. dragline) of spider silk proteins, and may lead to interesting biomaterials of desirable properties for biomedical applications. |
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