| Spider silk (Spider silk), a kind of natural protein fiber material, is well-known for its outstanding materials properties such as Strength, elastic and so on. Meanwhile, it is more and more used in research about medicine transport carrier and cell tissue engineering as a kind of biological macromolecule polymer, which has good biological compatibility and biological degradation. Orb-web spider can secrete six silk fibers, including minor ampullae gland silk (temporary capture silk) which is mainly used for reinforcing web and hunting, etc. Minor ampullae gland silk is similar to dragline silk, with the difference that it does not super contract in the water environment. And this characteristic makes it special application value. However, for inhabit of cannibalism, as well as the characteristics of the small amount of silk production, it is impossible to obtain silk fiber through the large-scale artificial breeding. With the rapid development of genetic engineering, it is only way to obtain spider silks by gene expression.In order to further study the structure and function of spider silk proteins, to reveal the mechanism of spider silk proteins into silk, explore to prepare bionic spider silk, this research aim to gain native spider silk protein product in Pichia pastoris system to realize large-scale fermentation production via intein splicing technology. For this reason, two fragments of MiSp gene are amplify from Araneous ventricosus MiSp repeat domain, which was depart into two sections with intern intron as boundary by PCR fused with his-tag and split-intein N/C end respectively, constructing4pairs expression cloning. It is expect to realize expressing, optimization, and purification to a high expression level in Pichia pastoris, then to get full-length protein product through spilt intein splicing in vitro.In the present study, because of independent recombination of two kind of intein and two kind of strains in P.pastoris, four pairs of cloning were construct, and one pair:GS115:P1-SXN, GS115:P2-SXC was realized expression and purification in P.pastoris.During the expression of GS115:P1-SXN, the control:BL21(DE3):P1-PKT was also expressed in E.coil. With the comparison of the SDS-PAGE and Western blotting result, it showed that soluble P1induced proteins expressed in different expression system could be obtained as the same size. Furthermore, the expression level and purification recovery efficiency was also calculated with2-3and5-7times more in GS115improved than that of BL21(DE3). Additionally, the expression level could be improved6-7times after optimizing the incubation and induction conditions of GS115.Besides these, spontaneous cleavage of split-intein was detected during expression of GS115: P2-SXC, so other3pairs of cloning should to be expressed to move forwards. In this research, P.pastoris expression system is more suitable for the native repetitive Gly/Ala-rich spider spidroin gene sequence expression than E.coil system.The data showed herein can be helpfully for the native full-length MiSp gene expression and large-scale exploitation of recombinant of spider silk proteins. |
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