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Use of the tetA-promoter in fed-batch cultivations: Repeated supply of anhydrotetracycline is necessary for production of tetrameric collagen prolyl 4-hydroxylase in Escherichia coli
Microbial Cell Factories volume 5, Article number: P77 (2006)
Human collagen prolyl 4-hydroxylase (C-P4H), an ER lumenal protein, is a key enzyme in the biosynthesis of collagens and consists of two different subunits forming an α2β2 tetramer. Heterologous cytoplasmic production of an active C-P4H in Escherichia coli using a bicistronic vector with the T5-lac and tet promoters and the strain Origami™ as a host was described earlier . Gene optimisation of the β subunit that is identical to protein disulfide isomerase and selection of the best induction conditions improved the obtained activity of recombinant C-P4H in shake flask cultivations further by a factor of 50 .
Although high amount of active C-P4H was obtained in shake flask cultures with long-time induction, the amount was low in fed-batch cultivations using the same induction strategy. Analysis of the mRNAs of the α and β subunits in fed-batch fermentations by Sandwich hybridisation revealed that single addition of the inducer anhydrotetracycline (aTc) leads to only transient induction of the α subunit, in contrast to IPTG which leads to a stable level of β mRNA.
Surprisingly, repeated induction with aTc led to a stable level of α mRNA and, consequently, to higher yields of the active C-P4H tetramers. The expression was strongly dependent on the cell density and the specific growth rate and provided best results if the culture was induced during the batch cultivation phase.
Our results indicate specific, so far non-described, features of the tet promoter based expression system. The repeated stepwise addition of aTc at higher cell densities might be of particular importance for other expression systems involving the tet A promoter and long expression time.
Neubauer A, Neubauer P, Myllyharju J: High-level production of human collagen prolyl 4-hydroxylase tetramer in Escherichia coli . Matrix Biol. 2005, 24: 59-68.
Niemitalo O, Neubauer A, Liebal U, Myllyharju J, Juffer AH, Neubauer P: Modelling of Translation of Human Protein Disulfide Isomerase in Escherichia coli . J Biotechnol. 2005, 120: 11-24. 10.1016/j.jbiotec.2005.05.028.
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Neubauer, A., Soini, J., Bollok, M. et al. Use of the tetA-promoter in fed-batch cultivations: Repeated supply of anhydrotetracycline is necessary for production of tetrameric collagen prolyl 4-hydroxylase in Escherichia coli. Microb Cell Fact 5, P77 (2006). https://doi.org/10.1186/1475-2859-5-S1-P77
- Shake Flask
- Protein Disulfide Isomerase
- Shake Flask Cultivation