From shake flasks to bioreactors: survival of E. coli cells harboring pGST-hPTH through auto-induction by controlling initial content of yeast extract |
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Authors: | Jia Lianghui Cheng Hairong Wang Hengwei Luo Huairong Yan Hua |
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Institution: | (1) State Key Laboratory of Phytochemistry and Plant Resources in West China, Center for Drug Screening and Research, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming, Yunnan, 650204, China;(2) Key Laboratory of Microbial Metabolism, the Ministry of Education, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China;(3) Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai, Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, China; |
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Abstract: | A high content of yeast extract in complex media can cause auto-induction of phage T7 RNA polymerase and the consequent expression
of recombinant protein in Escherichia coli BL21(DE3) during long-term cultivation. Our study demonstrated that the auto-induction of recombinant protein varied in different
vectors harboring heterologous genes. Trx, GST, and their fusion proteins such as GST–human parathyroid hormone (hPTH), expressed
by pET32a (+), were easily auto-induced by media containing a high content of yeast extract; however, rtPA was not easily
auto-induced when using pET22b (+), although both pET systems were under the control of T7lac promoter. Furthermore, the auto-induction of GST–hPTH may start within 1–2 h after inoculation in bioreactors, which is a
deficiency in the scale-up from shake flasks to bioreactors. Our results indicated that too much yeast extract in bioreactor
cultivations may be responsible for the early auto-induction of target proteins and consequent loss of cell viability and
plasmid instability. To achieve a satisfactory yield, host cells with both high cell viability and plasmid stability were
necessary for the starter cultures in shake flasks and pre-induction cultures in bioreactors. This could be achieved simply
by controlling the initial content of yeast extract and its subsequent supplementation. |
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