Prokaryotic Expression Of Maspl Protein From E.Australis

Most spiders can produce up to six silk fibers and a slime content, which all basing on protein. Due to their extraordinary mechanical and biochemical properties, spider silks have lots of potential application in various fields, such as textile, military, and biomedical engineering. Dragline silk as one kind of spider silk, called "biosteel", has received more and more attention. In contrast to silkworms, breeding spiders and harvesting their silk has been limited due to their cannibalistic nature. Therefore, obtaining mimic silks by gene engineering has been the focus for a long time.Dragline silk consists of two major polypeptides, referred to as major ampullate spidroin (MaSp)1and2. MaSpl is mainly composed of GGX iterated and poly-A module which is the key factor in determining the strength of dragline silk. The main component of MaSp2is GPGX which forms spiral structure and plays an important role in the elasticity property of dragline silk. Both of MaSpl and2contain non-repetitive N-terminal (NT, about130amino acid) and C-terminal region (CT, about110amino acid), and repetitive sequence (Rep).In order to obtain enough dragline silk protein, then produce the bionic silk fiber, the works we have done as follows:(1) Artificially synthesis NT, C1T, C2T, and4Rep gen sequences.(2) Mediated by Bsa I and BspM I restriction enzyme,the clones were constructed without inducing extra amino acids.(3) Optimizing expression conditions including temperature and vectors to improve yield of the silk protein; (4) Study the effect of the length of repeat modules in silk protein expression(5) Analysis the roles of NT and CT in the process of silk forming.All these researches have been successfully achieved:(1) According to the spider dragline silk protein gene sequence in GenBank (4Rep: AM490181; Cl:AJ973155; N:DQ059136; C2:AF350267), four cDNA sequences:NT, C1T, C2T, and4Rep, were artificially synthesized after codon optimization. The4Rep and C1T are from E.australis, while the NT and C2T are from A. trifasciata.(the information of NT of E. australis is limitied). The E. australis MaSpl poly-A contains12-15alanines which may show a better a mechanical prosperity.than others.(2) Mediated by Bsa I and BspM I restriction enzyme,4RepCT, NT4RepCT, NT8RepCT and NT12RepCT modulus were cloned in to the expression vectors without inducing extra amino acids.(3) The expression level of4RepC2T was successfully improved up to30mg/L by optimization of temperatures and vectors. In addition, recombinant NT4RepCT and NT8RepC2T proteins were also firstly produced with21and8.5mg/L expression level, respectively. The data revealed here indicate that thioredoxin(TRX) and S-tag of PET-32c(+) are useful for improving spider silk spidroin expression(4) The recombinant dragline silk proteins expression level went down as the increasing of the repeat length This phenomenon is mainly due to the high GC content and high content of Ala and Gly in4Rep module coding sequence, which leads to metabolism stress for E. coli in vivo, such as the lack of Ala-tRNA and Gly-tRNA.(5) Furthermore, the solubility of NT4RepC2is lower than that of4RepC2T by SDS-PAGE. That maybe partially atrributes to the pH sensitive NT module which is important in the forming of silk During the process of inclusion body purification for NT4RepC2T, the recombinant proteins formed a lot of Cys-based dimers, and after adding dithiothreitol (DTT, final concentration is5mmoL/L), dimers all disappeared. The CT domains of MaSps rely on Cysteins derived intra-dissulfide bonds to form stable dimers which promotes the orderly arrangement of upstream repeat regions (Ala and Gly-rich).In conclusion, the expression level of4RepC2T from E.australis was successfully improved by optimizating of temperature and vectors, and the role of NT and CT in spinning process.of silk protien were further analysed. These researches would be helpful to fully explore E. australis dragline silk proteins for mimic silk fibers with high performance.