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T7表达系统及自诱导蛋白产出策略已有1人参与
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    [ Last edited by zhangwei2491 on 2012-7-17 at 10:04 ] |
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楼主你的资源在哪里？》 给你一个连接：http://www.ncbi.nlm.nih.gov/pubmed/21523618 Appl Microbiol Biotechnol. 2011 May;90(4):1419-28. Epub 2011 Mar 10. From shake flasks to bioreactors: survival of E. coli cells harboring pGST-hPTH through auto-induction by controlling initial content of yeast extract. Jia L, Cheng H, Wang H, Luo H, Yan H. Source 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, Kunming, Yunnan, China. 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. PMID: 21523618 [PubMed - indexed for MEDLINE] |
2楼2012-07-17 09:18:05