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| Construction of a Vector Suitable for the Tandem Coexpression of Multiple Genes by a Single Plasmid |
| HE Zhang-hua1,2, WANG Yang2, ZHAO Jun2, LIU Xiao-jie2, ZHANG Li-hua2, WANG Dong2, SHI Ming-lei2, HUANG Fen1, YOU Ping1, ZHAO Zhi-hu2 |
1. College of Life Science, Shaanxi Normal University, Xi’an 710062, China;
2. Beijing Institute of Biotechnology, Beijing 100071, China |
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Abstract To develop an robust strategy for the tandem repeat co-expression of multiple genes in one plasmid, the point mutations were introduced into commercial expression vector pET-22b by the overlap PCR. After the mutation, the restriction enzyme site XbaⅠ was moved to the upstream of T7 promoter and a SpeⅠ site was introduced to the downstream of T7 terminator. The whole expression cassette including the T7 promoter, ribosome binding sites, multiple cloning site, T7 terminator were covered by the XbaⅠ-SpeⅠrestriction fragment. Restriction digestion and sequencing indicate that the mutated expression vector pET-m22b was constructed successfully. The four genes of different sources were choose for the coepxression by the pET-m22b. The SDS-PAGE shown that the four genes were highly co-expressed and without any affection on each other. The results indicated an efficient co-expression vector suitable for the tandem combination expression of multiple genes in single plasmid was constructed, which will make the co-expression of many genes more easy and will also greatly facilitate the expression and purification of multi-subunit protein complex, the rapid heterogenesis re-construction and optimization of biochemical pathways etc.
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Received: 20 August 2010
Published: 25 January 2011
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Cite this article:
HE Zhang-hua, WANG Yang, ZHAO Jun, LIU Xiao-jie, ZHANG Li-hua, WANG Dong, SHI Ming-lei, HUANG Fen, YOU Ping, ZHAO Zhi-hu. Construction of a Vector Suitable for the Tandem Coexpression of Multiple Genes by a Single Plasmid. China Biotechnology, 2011, 31(01): 40-45.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2011/V31/I01/40
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