Sequential and simultaneous statistical optimization by dynamic design of experiment for peptide overexpression in recombinant Escherichia coli |
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Authors: | Kwang-Min Lee Chang-Hoon Rhee Choong-Kyung Kang Jung-Hoe Kim |
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Institution: | (1) Cellular Metabolic Engineering Lab., Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong Yuseong-gu, 305-701 Daejeon, South Korea;(2) KoBioTech Co., Ltd., 713-12 Gojan-dong Nam-gu, 405-821 Inchen, South Korea |
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Abstract: | The production of recombinant anti-HIV peptide, T-20, in Escherichia coli was optimized by statistical experimental designs (successive designs with multifators) such as 24–1 fractional factorial, 23 full factorial, and 22 rotational central composite design in order. The effects of media compositions (glucose, NPK sources, MgSO4, and trace elements), induction level, induction timing (optical density at induction process), and induction duration (culture
time after induction) on T-20 production were studied by using a statistical response surface method. A series of iterative
experimental designs was employed to determine optimal fermentation conditions (media and process factors). Optimal ranges
characterized by %T-20 (proportion of pepttide to the total cell protein) were observed, narrowed down, and further investigated
to determine the optimal combination of culture conditions, which was as follows: 9, 6, 10, and 1 mL of glucose, NPK sources,
MgSO4, and trace elements, respectively, in a total of 100 mL of medium inducted at an OD of 0.55–0.75 with 0.7 mM isopropyl-β-d-thiogalactopyranoside in an induction duration of 4 h. Under these conditions, up to 14% of T-20 was obtained. This statistical
optimization allowed, the production of T-20 to be increased more than twofold (from 6 to 14%) within, a shorter induction
duration (from 6 to 4 h) at the shake-flask scale.
Coauthors. |
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Keywords: | Anti-HIV peptide statistical optimization dynamic experimental design |
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