Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli | |
Jiang Shan1,2; Liu Mei1; Wang Baojie1; Jiang Keyong1; Wang Lei1 | |
2011-11-01 | |
发表期刊 | CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY |
ISSN | 0254-4059 |
卷号 | 29期号:6页码:1249-1259 |
文章类型 | Article |
摘要 | 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, (NH(4))(2)SO(4) and KH(2)PO(4), 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 (NH(4))(2)SO(4) and 3.18 g/L KH(2)PO(4), 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 degrees 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.; 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, (NH(4))(2)SO(4) and KH(2)PO(4), 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 (NH(4))(2)SO(4) and 3.18 g/L KH(2)PO(4), 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 degrees 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. |
关键词 | Anti-lipopolysaccharide Factor Optimization Medium Composition Culture Conditions Statistical Methodology |
学科领域 | Marine & Freshwater Biology ; Oceanography |
DOI | 10.1007/s00343-011-0282-5 |
URL | 查看原文 |
收录类别 | SCI |
语种 | 英语 |
WOS记录号 | WOS:000299039400012 |
引用统计 | |
文献类型 | 期刊论文 |
条目标识符 | http://ir.qdio.ac.cn/handle/337002/12039 |
专题 | 海洋生物技术研发中心 |
作者单位 | 1.Chinese Acad Sci, R&D Ctr Marine Biotechnol, Inst Oceanol, Qingdao 266071, Peoples R China 2.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China |
第一作者单位 | 中国科学院海洋研究所 |
推荐引用方式 GB/T 7714 | Jiang Shan,Liu Mei,Wang Baojie,et al. Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli[J]. CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY,2011,29(6):1249-1259. |
APA | Jiang Shan,Liu Mei,Wang Baojie,Jiang Keyong,&Wang Lei.(2011).Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli.CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY,29(6),1249-1259. |
MLA | Jiang Shan,et al."Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli".CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY 29.6(2011):1249-1259. |
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