| The silk glands of Araneidae spider are in different forms and can secrete up to seven different spidroins,which constitute spider silks with different functions and different responsibilities in the process of spider growth,foraging,and reproduction.Spider silk has excellent mechanical properties such as tensile strength,extensibility,and toughness,as well as biocompatibility and biodegradability.It has a broad application prospect in areas of aerospace,materials science,tissue engineering,biomedicine,and so on,which makes spider silk a research hotspot in recent years.Despite the potential applications of spider silk,there are limitations in harvesting sufficient quantities of the silk proteins from spiders,owing to their aggressive behavior and territorial nature.Researchers turned to the production of recombinant spidroins and the preparation of artificial spider silk fibers to achieve the industrial production and wider application of spider silk fibers,and impressive progress has been made.Researchers have designed a variety of recombinant spidroins based on natural spider silk genes,expressed them in various expression systems,and then spun them through different spinning equipment and spinning methods to obtain high-performance artificial spider silk fibers.However,due to the large size of spider silk genes and high repetitiveness of gene sequences,the difficulties of obtaining spider silk genes and subsequent recombinant expression of spidroins are increased.And the research on the structure and function of spidroin and the complex and precise silk-forming environment in spider glands has not been completely decoded.Therefore,researchers haven't got artificial spider silks that fully mirrors the superior properties of natural spider silk so far.For these problems in spider silk research,we designed two sets of chimeric recombinant spidroins based on spider silk genes of two spiders:Euprosthenops australis and Araneus ventricosus,their non-repetitive regions used highly soluble N-terminal domain and C-terminal domain to ensure the recombinant protein's high solubility,repeat regions chose typical repetitive motifs from major ampullate spidroin(MaSp)or repetitive units from aciniform spidroin(AcSp),which endow high performance to the artificial spider silk fibers,and also explored different artificial spider silk spinning methods.Besides,we also obtained the non-repetitive terminal domain NT sequence and part of the repetitive region sequence of Araneus ventricosus MaSp1 through gene cloning,which fills the gaps in the Araneus ventricosus spidroin coding genes and provides material for the recombinant spidroin design and study.Study 1:Structure and biomimetic spinning study of NTnRepCT protein.At present,the performance of most artificially prepared spider silk fibers is far from comparable to that of natural spider silk,and the mass production and application of artificial spider silk fibers have not yet been realized.These have a lot to do with the low solubility of recombinant spidroins and the disadvantages of spinning methods.With the deepening research on spidroins,the roles played by the various domains of spidroins in the process of silk formation and their responses to the physical and chemical environment in the silk glands,such as pH reduction,ion concentration changes,shear force,and dehydration,showed that at least two prerequisites need to be met before it is possible to obtain biomimetic spider silk fibers with excellent material properties:first,the recombinant spidroin needs to be highly soluble and with high pH-sensitive;second,the spinning system needs to simulate the physical and chemical conditions in natural spider silk gland.Addressing these two key issues,we designed a series of chimeric recombinant spidroins(NTnRepCT),by selecting highly soluble terminal domains to ensure the highly soluble and pH-sensitive of the expressed recombinant spidroins,and designing a biomimetic spinning device by simulating the key conditions in the natural silk forming process to obtain biomimetic spider silk fibers with excellent performance.In this study,the chimeric recombinant spidroins are composed of two terminal domains on each side:N-terminal domain(NT) from Euprothenops australis MaSp1and C-terminal domain(CT) from Araneus ventricosus MiSp,and the core repetitive domain of the typical short repetitive motif(poly A and Gly-rich motif)with different lengths from E.australis MaSp1.Highly soluble NT2RepCT and NT2*2RepCT proteins(both above 500 mg/m L) were obtained through Escherichia coli expression and affinity purification,which proved that the highly soluble NT and CT play a vital role in the soluble expression and high solubility of spidroins,and it is the first report in spider silk research that the solubility of recombinant spidroins reached or even exceeded the storage concentration of natural spidroin in silk gland.Circular dichroism(CD) analysis of both proteins in solution state showed the recombinant spidroin mainly exists in the form of?-helix secondary structure,and the mass spectrometry(MS) analysis and Cryo-electron microscopy(Cryo-EM)observation showed NT2RepCT has high pH-sensitivity.By designing a novel and ingenious biomimetic spinning apparatus,the high-concentration spidroin spinning stock solution can be stably spun into the toughest as-spun spider silk fiber(up to it reported,45 MJ/m3).Using Fourier infrared spectroscopy(FTIR)to analyze the structural composition of spidroin before and after spinning,the results showed that in the silk fiber,the ?-helical structure of the biomimetic spinning spidroin was reduced,while the ?-sheet increased,which was following the structural change trend during the natural spider silk formation.In addition,there was no involvement of organic reagents during the whole process from the recombinant spidroins expression to protein purification to biomimetic spinning,which increases the safety and applicability of silk fiber in biomedicine applications.Study 2:Structure and artificial spinning study of iNnRC protein.Though the different types of spider silks have different performances and undertake different functions,the structural patterns of spidroins that make up different spider silks are the same,that is,they all have a non-repetitive N-terminal domain(NT),a middle repetitive domain(REP),and a non-repetitive C-terminal domain(CT).NT and CT are usually short in length and have high sequence conservation among spidroins from different spiders.These two domains are important for the high concentration storage of spidroins in silk gland and play a significant role in silk assembly initiating.While the REP domain,which could occupy up to more than 90%of the spidroins composition,has low sequence conservation.Different spidroins are composed of repetitive modules of different lengths and numbers,the number of repetitions can even be up to hundreds of times,and the combination of these different repetitive modules determines the unique material properties of different spider silks.To obtain high-performance artificial spider silk materials,we selected different numbers of repeating units of the highly soluble repetitive domain from aciniform spidroin flanked by the highly soluble N-terminal domain and C-terminal domain from minor ampullate spidroin to obtain a series of high-yield and highly soluble recombinant spidroins.Through different spinning methods to prepare artificial spider silk fibers with high performance,and to explore the relationship between spidroin size and silk performance.In this study,we constructed a series of chimeric recombinant spidroins(iNnRC)based on two types of spider silk genes from Araneus ventricosus,that are the non-repetitive terminal domains of NT and CT from minor ampullate spidroin(MiSp),and 1?7 repetitive unit R from the repetitive domain of aciniform spidroin(AcSp).Seven chimeric recombinant spidroins all successfully expressed in the form of inclusion body in Escherichia coli cells,purified by urea,and obtained at high-yield and high-purity.Artificial silk fibers can be prepared by using three spinning methods:biomimetic spinning,hand-drawing,and HFIP wet spinning.Circular dichroism(CD) analysis of spidroins in solution state showed the?-helix structure gradually loses its dominance as pH decreases.Fourier infrared spectroscopy(FTIR) analysis of the silk fibers structure showed ?-sheet structure becomes the main structural component after silk fiber for-mation,which accounts for more than 40%.The tensile performance test results revealed that the silk fiber prepared by hand-drawing has the best overall performance,thereinto,the strength of iN7RC silk fiber is 345 MPa,while its extensibility is up to 38%.The results showed a positive correlation between the performance of silk fiber and the number of repeating units in recombinant spidroin,which also refers to the protein size.These provide new research materials and approaches for the preparation of artificial spider silk fibers.Study 3:The study of MaSp1 terminal domains.Major ampullate silk serves as supporting radii and mainframe of spider web,and also the spider's lifeline(also known as dragline silk),and it has become a hotspot in spider silk research due to its extraordinary mechanical properties and easy isolation from the web.Major ampullate silk consists of major ampullate spidroin(MaSp),which include two components MaSp1 and MaSp2,and the molecular weight of MaSp is relatively high,up to 250?350 kDa.Studies have shown that the NT domain of the main ampullary gland silk protein ensures the rapid filamentation of MaSp through a three-step dimerization mechanism in a pH-dependent manner,while its CT domain exists as a dimer to trigger and regulate the silk formation process.Therefore,obtaining more coding genes and more structural and functional information of NT and CT from different spider species are quite important for the production of artificial spider silk with high performance.In this study,the NT and CT of Araneus ventricosus MaSp1 were focused on,and the complete coding sequence A.v.NTMa1 and part of the repetitive domain sequence of Araneus ventricosus MaSp1 were first obtained by anchored PCR,which filled the vacancy of Araneus ventricosus spider silk coding genes.The alignment of the obtained NT sequence and NT sequences from known MaSps of other spider species showed that the NT sequence is highly conservative.The recombinant protein of NT and CT of A.ventricosus MaSp1 was successfully expressed in Escherichia coli cells,and the purified protein was obtained with high purity.The structural composition analysis of the two proteins in the solution state by circular dichroism(CD) revealed that both proteins were consistent with their secondary structure predictions,in which the?-helix struc-ture was dominant.The results provide data support for the high-level structure and function analysis of the NT and CT terminal domains of MaSp1,enrich the known spider silk coding genes,and provide research material for developing potential new uses of spidroin materials,such as soluble tags,and so on.It also laid the foundation for the study of the structure and silk formation mechanism of Araneus ventricosus MaSp1.The thesis carried out systematic research on the design and expression of recombinant spidroin,silk fiber preparation,and spider silk gene acquisition.For the first time,the recombinant spidroin spinning stock that reaches natural spidroin storage concen-tration was obtained,a convenient and efficient biomimetic spinning device was designed,and through which successfully obtained as-spun spider silk fiber with the high-est toughness.The results of the research conducted here verify the close correlation between the composition and size of spidroin and the performance of the spider silk,fill in the gap of the spider silk genes,provide new thoughts to the design and expression of the recombinant spider silk protein and the spinning preparation of silk fiber,offer abundant raw materials for the application research of different spider silk protein materials,and also serve as genetic support for the study of spider silk gene evolution and spider genetics. |
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