title: From shake flasks to bioreactors: survival of E. coli cells harboring pGST-hPTH through auto-induction by controlling initial content of yeast extract.
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.
authors.affiliation: 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.
authors.forename: Lianghui
authors.initials: L
authors.lastname: Jia
authors.forename: Hairong
authors.initials: H
authors.lastname: Cheng
authors.forename: Hengwei
authors.initials: H
authors.lastname: Wang
authors.forename: Huairong
authors.initials: H
authors.lastname: Luo
authors.forename: Hua
authors.initials: H
authors.lastname: Yan
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D003470:Culture Media / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D050296:Microbial Viability; D010281:Parathyroid Hormone; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; C470125:PTH protein, human; D010281:Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase