Repeat Modules In GeneEngineering Expressed Biomimetic Spider Protein Determine Silk Fiber Properties

As a kind of protein material, spider silk has excellent material properties that can be used in aerospace, biomedical engineering, military projects such as flak suit. Although spider silk cannot be produced by simply raising spider like silkworm because of spider internecine nature instinct, scientists have tried to express spider silk gene in goat, plant, insect cell after the end of 20th century. However, the difficulties of overall length gene expression problem and low protein productivity have not been solved nowadays.The gene of spider silk is mainly composed of N non-repeat terminal, C non-repeat terminal and many repeat modules between N-terminal and C-terminal. At present, the research of spider silk focus on aspects as followed:1, Constructing library to retrieve the overall length of spider silks from different species and glands; 2, Understanding spider silk protein into fiber mechanisms, mainly referring to the functions of N-terminal, C-terminal and middle repeat sequences in the process spider silk proteins transfer into fibers, also including proteins the change of proteins secondary structure in self-assembly. 3, Application of spider silk in biomedical engineering relate in composite material, drug delivery and so on. At the present, research of spider silk become more and more hot. Many research papers were continuously published in 2010 Nature magazine related the mechanism of spider silk protein transferring to protein fiber. More and more people pay close attention to application area of spider silk. Thomas R. Scheibel selectively embeded and released drug by self-assemble character of spider silk protein, formed capsules or spheres to apply in Drug Dlievery System.Composite material constituted by spider silk protein and tissue engineering material become better histocompatibility and degradable material which will be used in the wide range of alternative tissue and sutures.In some papers reported at present show that N-terminal non-repeat domain and C-terminal non-repeat domain are both very important in the process of silk self-assembly,The N-terminal domain comprises a secretion signal and is sensitive to pH. The C-terminal domainwas implicated in the control of solubility and fibre formation .Comparing to the N-terminal domain and the C-terminal domain, repeat domain have lower homology in different spider species and spider silks. Large molecular weight and complicated structure increase the research problem of repeat domain. Functions of repeat domain in the fiber formation process remain unassigned.We research the material property of silk fiber with different number of repeat domain in molecular biology and matcrialogy.A 693bp partial fragment of spider flagelliform repeat domain was isolated from Araneus ventricosus gene library and this fragment contain an intact repeat domain of flagelliform silk gene. The gene was then optimized as E.coli codon and repeated 1-4 times respectively, meanwhile added the no-repeat N-terminal and C-terminal domains to the flank of the repeat fragments. Four recombination genes fused with intein and CBD (chitin binding domain) were expressed in IMPACT (Intein-Mediated Purification with an Affinity Chitin-binding Tag) system and purificated by affinity chromatography using chitin beads. DTT induced N-terminal cleavage of modified intein.Four target proteins N-Sc1/2/3/4-C could be elution from chitin beads without any exogenousamino acid residues.Dialysis the pure proteins in pH 7.0 deion water respectively. The proteins after freeze drying are solubilized in 1, 1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 20%(wt/vol) for extrusion protein solutions into fibers by wet-spinning.Secondary structure in four kinds of biomimetic spider silks could be anlysised by Raman test. The structure of GPGGX in Repeat modules formβ-turn spiral which benefits the elasticity of spider silk. Protein secondary structure ofα-helix,β-sheet and random coil are mainly stored in N-terminal and C-terminal.The test results show there are not too many differences.The machenical propertiesof fibers with different repeat modulescould be checkouted by Stress-strain test. We can find flagilliform repeat domain mainly determine the extensibility of biomimetic spider silk.All of my researches want to explain the number of spider flagelliform repeatdomains determine the fiber properties in the process of protein into fiber. We could find relationships between the amino acid sequences and the secondary structure of spider flagelliformrepeat domain. At the sametime, an efficiency system will be established to producegene engineering expressed biomimetic spider silk.