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young1983木虫 (正式写手)
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[求助]
求助芽孢杆菌载体pHT254 已有1人参与
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求助芽孢杆菌载体pHT254 发自小木虫IOS客户端 |
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林科院林化所招收材料与化工专业硕士，坐标南京
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2楼2018-11-05 14:30:43
【答案】应助回帖
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1. 为大肠杆菌/枯草芽孢杆菌穿梭质粒。 2. IPTG诱导，控制目的蛋白枯草芽孢杆菌胞内表达。 2. pHT254与pHT01质粒骨架相同。 3. pHT254启动子是Pgrace100, 目的蛋白在枯草芽孢杆菌中表达较高，在大肠杆菌中表达较低（背景）。 4. pHT01启动子是Pgrace01（或称Pgrace）, 相比pHT254启动子系统，目的蛋白在枯草芽孢杆菌中表达低些，在大肠杆菌中表达高一些。 参考文献：Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli。 Phan et al. Microbial Cell Factories (2015) 14:72 Abstract Background: In general, fusion of recombinant genes to strong inducible promoters allowing intracellular expression in Bacillus subtilis is a two-step process. The ligation products are transformed into Escherichia coli, followed by identification of the correct plasmid, and this plasmid is subsequently transformed into B. subtilis. This raises the problem that basal level of expression of the recombinant gene could be harmful for E. coli cells. Based on the Pgrac promoter, we optimized the UP element, the -35, 15, -10 and the +1 region to enhance the promoter activity in B. subtilis after induction. However, detailed investigations for a promoter to develop expression vectors that allows high protein production levels in B. subtilis and a relatively low basal expression levels in E. coli has not been studied yet. Results: We screened the previously constructed library of E. coli ?B. subtilis shuttle vectors for high level expression in B. subtilis and low basal level in E. coli. Promoter Pgrac100 turned out to meet these criteria, in which ?galactosidase expression level of Pgrac100-bgaB is about 9.2 times higher than Pgrac01-bgaB in B. subtilis and the ratio of those in induced B. subtilis over un-induced E. coli from Pgrac100-bgaB is 1.3 times higher than Pgrac01-bgaB. Similarly, GFP expression level of Pgrac100-gfp is about 27 times higher than that of Pgrac01-gfp and the ratio from Pgrac100-gfp is 35.5 times higher than Pgrac01-gfp. This promoter was used as a basis for the construction of three novel vectors, pHT253 (His-tag-MCS), pHT254 (MCS-His-tag) and pHT255 (MCS-Strep-tag). Expression of the reporter proteins BgaB and GFP using these expression vectors in B. subtilis at a low IPTG concentration were measured and the fusion proteins could be purified easily in a single step by using Strep-Tactin or IMAC-Ni columns. Conclusions: This paper describes the construction and analysis of an IPTG-inducible expression vector termed Pgrac100 for the high level production of intracellular recombinant proteins in B. subtilis and a relatively low basal expression level in E. coli. Based on this vector, the derivative vectors, Pgrac100-His-tag-MCS, Pgrac100-MCS-His-tag and Pgrac100-MCS-Strep-tag have been constructed. |
3楼2018-11-05 15:54:27
4楼2018-11-05 15:56:33
young1983
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5楼2018-11-05 21:56:57
swordhang
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