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Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli

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Abstract

Anti-lipopolysaccharide factors (ALFs) are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Escherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, (NH4)2SO4 and KH2PO4, were further optimized via the central composite design (CCD). Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside (IPTG) concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L (NH4)2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7°C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% (without optimization) to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 10.5% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.

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Authors and Affiliations

  1. R&D Center of Marine Biotechnology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China

    Shan Jiang  (姜姗), Mei Liu  (刘梅), Baojie Wang  (王宝杰), Keyong Jiang  (蒋克勇) & Lei Wang  (王雷)

  2. Graduate University of Chinese Academy of Sciences, Beijing, 100049, China

    Shan Jiang  (姜姗)

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  1. Shan Jiang  (姜姗)
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  2. Mei Liu  (刘梅)
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  3. Baojie Wang  (王宝杰)
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  4. Keyong Jiang  (蒋克勇)
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  5. Lei Wang  (王雷)
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Correspondence to Lei Wang  (王雷).

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Supported by the National High Technology Research and Development Program of China (863 Program) (No. 2006AA100311)

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Jiang, S., Liu, M., Wang, B. et al. Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli . Chin. J. Ocean. Limnol. 29, 1249–1259 (2011). https://doi.org/10.1007/s00343-011-0282-5

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  • DOI: https://doi.org/10.1007/s00343-011-0282-5

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