Production And Characterization Of Hybrid Aciniform Silk

Due to the remarkable mechanical properties, antimicrobial properties and no influence on biocompatibility and immunological response, spider silk hence is considered as most promising biomaterials. Spider silks have been the focus of research for decades, but its low availability due to the cannibalism of most spiders, large-scale production of spider silk is not possible. Therefore, it is essential to exploit biotechnology to clone the gene and express recombinant gene in vitro via various host cell. Electrospinning has been used to producing core–shell functionalized nanofibres. However, the organic solvents had unfavorable effects on the native structure of active protein domain, resulting in the deactivation of functional protein during the encapsulation procedure. Fiber produces from micro fluid device miming the natural spinning process of spider is believe to be a promising biomaterial.Here three proteins with different molecular weight, including recombinant silk protein C8CT(38 KD), maltose binding protein(MBPHC, 40 KD), thioredoxin(THC, 12 KD) were employed and fluorescence-labeled for testing easier. Proteins mixed equally with recombinant AcSp proteins W2 CT in 50 mM K3PO4 pH 7.5 and fibers were generated by means of hand-drawing respectively to spin hybrid silks. Results from the coommassie blue staining gel and fluorescence scanning image prove the exogenous protein with different origin and molecular weigh can be incorporated into the fibers instead of attachment to the surface randomLy, which were also supported by the fibers image taken from fluorescence microscope. Although mechanical property test shows hybrid silk slightly reducing stress comparing to fiber from W2 CT, Flush washing, two days immersing of the fibers into physic buffer and lower concentration urea corrosion experiments shows us the exogenous proteins are stable and uniform in the fibers without burst release in two days long. The results here imply that the protein originated-drugs with molecular weight similar to the three model proteins may be incorporated and stable in the hybrid fibers by manual hand drawing way. We believe this work established the platform for further producing functional hybrid fibers with potential application via micro fluid device miming the natural spinning process of spider.Meanwhile, to provide technical support for spider silk functional modification, and develop functionalized silk materials which present whole folded protein domains via fused protein. We tried to explore a simply and efficient functionalized platform via intein trans-splicing. Small ubiquitin-related modifier protein(SUMO) was fused to the recombinant spider silk protein(W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, which results in a protein SUMOW2 CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT(W). SUMO protease digestion showed positive results on the fibers, which indicates the SUMO protein keeps its native conformation and bioactive. Above all, the success of this work provides a technical support for spider silk high simply and efficient functionalized modification.Chimeric fibers carrier exgenenous protein by mixing and fusing protein together leads to a decrease of tensile strength. Due to its controlled mechanical properties, hybrid silks hold a great potential as specific biomaterials. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4 CT and W2C8 CT, with ligation of MaSp repetitive modules(C) with AcSp modules(W) by intein trans-splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K3PO4 pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance compared with fibers from W2CT(W fiber) and mixture of W2CT/C8CT(MHF8 fibers). Fibers from chimeric protein W2C4CT(HFH4) have improved mechanical properties than W fibers, however, with more C modules W2C8 CT fibers(HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans-splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks.The facility of adoption intein in fiber functionalzation and mechanical property improvement is obviously. But the splicing efficient is relied to the neighbouring extein, which hinders the application of split intein. While the previous established Kana selection system is not suitable in spilt intein directed evolution selection.To develop a platform for split intein in vitro function selection, we proposed a DNA display system. In this system, streptavidin-fused intein proteins are linked with their encoding biotinylated gene in vitro compartments and can be selected via the tag labeled by trans-splicing. By introducing western blot and DNA electrophoresis analysis system, we found this system can express the designed gene(IRC and IRCM)(~500aa). Besides intein from positive control(IRC) still hold it splicing function,we achieved DNA-protein conjugates with highly efficient(>95%). Furthermore, we successfully enriched a positive gene(IRC) from the negative gene(IRCM) by a factor of 2-5 fold per round on anti-flag m2 beads(R0=1:10). Thus, DNA display should be useful for rapidly screening or evolving intein based on affinity selection.Our research provided a reasonable method for producing hybrid silk protein by mixing wrapping silk protein with exogenous protein or fusing wrapping silk protein with exogenous protein via intein trans-splicing result in spider silk with extra property. The consequential fiber with decrease in tensile strength can be overcame by producing hybrid silk from wrapping silk module and dragline silk module. The following split intein selection system provide a new strategy for split intein directed evolution which will expand the application field of split intein. Above all, our research develop a platform for protein functionalization or mechanical property improvement, when using recombinant spider silk as biomaterials.