| With the development of genetic engineering, more and more recombinant therapeutic protein products have appeared and their market prospect is also more and more broad. Recombinant therapeutic protein production via heterologous expression systems is the crucial content and research hotspot of the modern biotechnology industry. Among the many systems available for heterologous protein production, the gram-negative bacterium Escherichia coli remains one of the most attractive because of its ability to grow rapidly and at high density on inexpensive substrates, its well-characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains; and its expression of exogenous protein is much higher than the other protein expression system, the amount of target protein expression of bacteria is even more than 30% of the total proteins. However, some recombinant proteins especially these proteins with muti-disulfide bond may exist mainly as inclusion bodies when they were expressed in E. coli. Although these proteins can be successfully expressed by Mammalian cell, the low yield, high cost, long productive cycle for mammalian cells seriously limits the popularization and application of genetically engineered products. So improving the E. coli expression system which can be used to express muti-disulfide bond proteins successfully is a challenging issue. We had selected the Aspergillus phytase which own multi-disulfide bonds as the target, explored and improved the approach for the prokaryotic expression of phytase. Based on the Aspergillus phyA gene which was early screened in our laboratory, we took two strategies to expression of phytase. One way is to transfer the recombine protein from reduced cytoplasm to oxidative periplasm by adding different signal sequences; the other method is using E. coli mutant strains with oxidative cytoplasm to express this enzyme. In this article, we not only tested the available of the two strategies, but also optimized the best expression conditions of the engineering strains. The following results were achieved:1. Oxidative cytoplasm can significantly enhance the catalytic activity of phytase. Two mutated strains with the oxidative cytoplasm, Rosetta-gami(DE3) and TransB(DE3), were chosen as the host strains. Since the oxidized cytoplasm environment of mutant strains and oxidation state of the thioredoxin family, the mutant strains are favorable for the formation of disulfide bond. Our results showed that the catalytic activity of phytase has been improved under the usual cultivate-induced condition. The activities of PhyA-Rosetta-gami and PhyA-TransB were improved 234% and 220%, respectively.2. Transport to the periplasm is favorable for improving the catalytic activity of phytase. Four different signal sequences (SSPelB, SSDsbA, SSPhoA, and SSDsbC) were linked to the N-terminal of the phytase, which have been reported to tanslocate the recombine protein efficiently to the periplasm. And periplasm not only has oxidizing cytoplasm but contains enzymes for disulfide bond formation (Dsb system). The activities of phytase expressed with the signal peptide are all higher than that of phytase expressed in the usual strains. Adding SSPelB, the phytase activity increased 164%, while the SSDsbA-PhyA is the highest one improved 246%. The SSPhoA-PhyA and SSDsbC-PhyA improved 225% and 186%, respectively.3. Explore the optimal cultivation-induction conditions. We have optimized cultivation-induction conditions for the catalytic activity of phytase which expressed in periplasm and oxidative cytoplasm. The results showed that concentration of IPTG affected the activity of phytase significantly, while temperature has little effect. In general, the highest catalytic activities usually appear under the condition of 0.25mM IPTG; the impact of temperature on the phytase activity is complex, and the best induced temperature is relatively dispersed, distributed in 25℃, 28℃, 34℃and 37℃.4. Optimization of fermentation is favorable for improving the catalytic activity of phytase. In the periplasm Transportation, the most efficient one is SSDsbA-PhyA which make the catalytic activity improve 525% compared with that of phytase without the signal sequence under its optimum expression conditions (28℃/0.25mM IPTG). As to the oxidative cytoplasm expression, the most efficient one is PhyA-Rosetta-gami and its catalytic activity of phytase increased 542% compared with the reductive control under its optimum expression conditions (34℃/0.50mM IPTG).
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