Expression,Purification Of Recombinant Spidroins And Characterization Of Silk Fibers

Spider silk with excellent mechanical properties has wide application prospects in biomedical,military and aerospace fields.At present,it has become the mainstream to express spider silk protein by genetic engineering and obtain recombinant silk fiber by spinning.However,there are many repeat sequences in spider silk protein,the mechanism of natural silk formation has not been thoroughly studied,and the spinning process is quite different from the process of natural silk formation.These factors make it difficult for bionic spider silk to replicate all the characteristics of natural spider silk.To further explore the recombinant silk fibers that are closer to the properties of natural spider silk,we first selected the chimeric spidroin(FAc)in the laboratory as the research object,which is composed of one repetitive module of flagelliform spidroins(Flag)from Araneus ventricosus and one complete repetitive module of aciniform spidroins 1(Ac Sp 1)from Argiope trifasciata.The young's modulus of this chimeric spidroin exceeded that of natural aciniform silk.The stress was about 1/3 of that of natural aciniform silk.But the strain and toughness were low.There have been reports indicating that increasing the number of repetition regions can improve the mechanical properties of spider silk.However,most of the reports were about the repeated modules of a certain type of spider silk,and there were few reports of combined repetitive modules of different types of spidroins.Therefore,we combined constructed chimeric expression plasmids p EHS-FAc-FAc and p EHS-FAcFAc which contains 2 or 3 repeats of FAc modules.It was expressed in Escherichia coli and purified by Ni-NTA column.The FAc-FAc fibers were obtained by manual-drawing,and scanning electron microscope(SEM)showed that the fiber diameter was uniform,and the appearance was smooth.Then the mechanical properties of the silk fibers were tested and compared with the chimera spidroin FAc.It was found that the mechanical properties of the spider silk fibers were improved to varying degrees by superimposing repeated modules,especially the toughness was increased by nearly three multiples,reaching an average of 122.28 MPa.Young's modulus was still comparable to that of natural aciniform silk.The structural analysis showed that the main secondary structure change from ?-helix to ?-sheet during the process from spider silk protein solution to spider silk fiber was consistent with the results in previous reports.But p EHS-FAc-FAc-FAc could not be successfully expressed in Escherichia coli due to its high molecular weight(? k Da)and repetitive sequences.Trans-splicing will be used for FAc-FAc-FAc ligation in the future study.It was proved that the superposition of repeated modules could effectively improve the mechanical properties of silk fibers without the non-repeating Nterminal domain(NT)and C-terminal domain(CT),providing a new idea for designing highperformance chimeric spider silk fibers to a certain extent.At the same time,in order to further expand the application of functional spider silk fiber.In this study,the red fluorescent protein(RFP)was fused to the upstream of the chimeric spidroins(NRC and NRRC)by genetic engineering to obtain functional bionic spider silk.NRC or NRRC chimeric spidroins contained one or two repeat unit(s)of pyriform spidroin(Py Sp)from Araneus ventricosus flanking by NT and CT of minor ampullate spidroin(Mi Sp),which exhibited remarkable tensile strength.It was expressed by Escherichia coli and target proteins RFP-NRC and RFP-NRRC were obtained by denaturation and purification.Silk fibers with strong red fluorescence were obtained by wet spinning and the relationship between the water content of coagulating bath and the mechanical properties of the silk fiber was explored during wet spinning.The results showed that the higher the water content in a certain range,the better the comprehensive properties of the fiber will be.Compared with the silk fiber without red fluorescent protein,the stress and young's modulus of spider silk fibers were improved,and the most prominent improvement was the toughness,which reached177.56±33.96 MPa.The results showed that the obtained red fluorescent spider silk fiber still maintained excellent mechanical properties,and the fusion of RFP had little effect on the mechanical properties and secondary structure of the silk fibers.The functional fluorescent spider silk material is expected to be used for positioning and tracking,and combined with the antibacterial properties of spider silk itself,the functional spider silk fiber can be more widely used in biomedical and other fields,providing support for functional and high-performance spider silk fiber.In conclusion,high-performance spider silk fibers were obtained by superimposing repeated modules of spider silk proteins,providing theoretical support for the future application of highperformance chimeric spider silk.In addition,the high-performance red fluorescent spider silk fiber was successfully obtained by fusing red fluorescent protein with spider silk protein,which laid a foundation for further research and application of fluorescent spider silk fiber.