The Research Of MiSp Modules

Spider silk is an ideal super material due to its extraordinary mechanical properties compared to other available materials. However, mass production of spider silk from nature is still impossible because the farming of spiders is limited by their cannibalistic and territorial behavior. Thus, producing spider silk by recombinant biotechnology is one of the most promising alternatives. Before achieving this goal, besides the special express host which can support the full length expressiong of high molecular weight spider silk proteins, one needs to understand the molecular structures, self-assembly mechanism and fiber formation of spider silk proteins, which are affected by solvent environment, shear and elongational forces.The full-length MiSp protein contains non-repetitive N-terminal (NT) and C-terminal region (CT), and repetitive region (R). NT and CT are highly conserved which are sensitive to the environment changes in vivo and help the spidroin change into solid fibers, and the R domain is mainly relative to the properties. Sodium cholide is a rich composition in the silk gland, and it is thought that NaCl can play a very important role on the fibers formation.In this research, we studied the secondary structures of different MiSp modules under different pH, and also assembly and fiber formation under different ion and pH to discover the mechanisms of spider silk formation. The works we have mainly done are as follows:(1) Based on the full-length MiSp, recombinant clones are constructed, including R1R2、R1SR2、R1R2CT、R1SR2CT、NTR1R2CT、NTR1SR2CT which were verified bysequencing;(2) All clones were expressed in Rosetta2(DE3);(3) Recombinant proteins were puried by Ni-NTA with high purification;(4) CD spectrum was recorded at room temperature under different pH to characterize the secondary structures;(5) SEM was also applied to observe the protein assemble and fiber formation under different pH and ionic condition, which can give clues to the proteins-silk formation mechanism research.All these researches have been successfully finished:(1) Mediated by Bsa Ⅰ restriction enzyme, R1R2、R1SR2、R1R2CT、R1SR2CT、 NTR1R2CT、NTR1SR2CT modules were constructed successfully and expressed without inducing extra amino acids.(2) Rosetta2(DE3) host strains are applied to enhance the expression of the recombinant proteins that contain codons rarely used in E. coli. These strains supply tRNAs for7rare codones (AGA, AGG, AUA, CUA, GGA, CCC, and CGG) on a compatible chloramphenicol-resistant plasm id.(3) Due to the amino acids composition and structure, recombinant proteins with spacer have low purity after Ni-NTA purification; protein purity could be higher with increasing the imidazole concentration of wash buffer.(4) Because of the existence of the spacer, recombinant proteins R1SR2、R1SR2CT、 NTR1SR2CT are not stable, the CD signal is not very obvious. CD spectrum of six recombinant proteins are different:CD spectra at room temperature showed R1R2has similar secondary structure, random coil under pH7.5,6.5and5.5, and the predominant secondary structure of R1R2CT is α-helix. S, NT and CT three domains structure are related to the spiral structure. NTR1R2CT Spectra is the most obvious, with negative peak at195nm under pH7.5, indicating the random coil structure, with the pH decreasing to5.5, the random coil disappear, all the proteins fold into helix structure. NT and CT can promote the secondary structure changes from the random coil to helix or sheet under pH7.5to5.5. The whole data show the secondary structure for R1R2is random coil and α-helix for R1R2CT, in which CT can play an important role on stabilizing R1R2secondary structures.(5) In this study, SEM is applied to characterize the protein aggregation and fiber formation under different pH and ionic environment after250r/min shaking culture under37℃for12hours. SEM data, applied to observe the assemble and fiber formation under different pH with or without NaCl, indicated at pH5.5R1R2and R1R2CT both could form fibers spontaneously, but R1R2CT fibers are smooth and similar to native spider silks while R1R2fibers show crude fiber belt morphology. Furthermore, NaCI can give birth to fibers with crude morphology. Under pH5.5, R1R2and R1R2CT form fibers and NaCl can affect negatively fiber morphology.In conclusion, this study, including various tests under the conditions of different ions and pH values, is about to characterize the secondary structure and fiber-formation. In future, we need to increase the shear force to explore protein aggregation and spinning mechanism. The results obtained here can give clues to the proteins-silk formation mechanism research and be helpful to biomimic spider silks.