Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli
文献类型：期刊论文
| 作者 | Wlad, H; Ballagi, A; Bouakaz, L; Gu, ZY; Janson, JC |
| 刊名 | PROTEIN EXPRESSION AND PURIFICATION
 |
| 出版日期 | 2001-07-01 |
| 卷号 | 22期号:2页码:325-329 |
| 关键词 | mass-transport limitation biosensor technology bacterial expression interaction kinetics chain antibodies proteins |
| ISSN号 | 1046-5928 |
| 其他题名 | Protein Expr. Purif. |
| 中文摘要 | We report a rapid, large-scale process for the purification of a recombinant Fab fragment specific for the tobacco mosaic virus coat protein (Fab57P). The fragment is expressed periplasmically in Escherichia coli. The expression level was optimized in 0.3-L fermenters. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degreesC), (2) combined use of two beta -lactam antibiotics (carbenicillin and ampicillin), (3) IPTG concentration 0.1 mM, (4) regulated pH 7.2, (5) 17-h induction time, and (6) conditions that reduce mechanical stress. Optimized large-scale fermentations were done in 15- and 300-L capacity fermenters. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the elude E. coli homogenate was directly applied, without centrifugation, to an SP Sepharose Big Beads column. The recombinant Fab fragment was eluted as a single peak in a sodium chloride gradient. The fragment was further purified by affinity adsorption to a column packed with Epoxy-activated Sepharose 6B to which the antigen peptide NH2-CGS YNR GSF SQS SGLV-CONH2 had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%. (C) 2001 Academic Press. |
| 英文摘要 | We report a rapid, large-scale process for the purification of a recombinant Fab fragment specific for the tobacco mosaic virus coat protein (Fab57P). The fragment is expressed periplasmically in Escherichia coli. The expression level was optimized in 0.3-L fermenters. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degreesC), (2) combined use of two beta -lactam antibiotics (carbenicillin and ampicillin), (3) IPTG concentration 0.1 mM, (4) regulated pH 7.2, (5) 17-h induction time, and (6) conditions that reduce mechanical stress. Optimized large-scale fermentations were done in 15- and 300-L capacity fermenters. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the elude E. coli homogenate was directly applied, without centrifugation, to an SP Sepharose Big Beads column. The recombinant Fab fragment was eluted as a single peak in a sodium chloride gradient. The fragment was further purified by affinity adsorption to a column packed with Epoxy-activated Sepharose 6B to which the antigen peptide NH2-CGS YNR GSF SQS SGLV-CONH2 had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%. (C) 2001 Academic Press. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 研究领域[WOS] | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 关键词[WOS] | MASS-TRANSPORT LIMITATION ; BIOSENSOR TECHNOLOGY ; BACTERIAL EXPRESSION ; INTERACTION KINETICS ; CHAIN ; ANTIBODIES ; PROTEINS |
| 收录类别 | SCI |
| 原文出处 | |
| 语种 | 英语 |
| WOS记录号 | WOS:000169703900020 |
| 公开日期 | 2013-11-14 |
| 版本 | 出版稿 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/5782]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 作者单位 | 1.Uppsala Biomed Ctr, Ctr Surface Biotechnol, SE-75123 Uppsala, Sweden 2.Uppsala Biomed Ctr, Dept Cell & Mol Biol, SE-75124 Uppsala, Sweden 3.Chinese Acad Sci, Inst Chem Met, State Key Lab Biochem Engn, Beijing, Peoples R China |
| 推荐引用方式 GB/T 7714 | Wlad, H,Ballagi, A,Bouakaz, L,et al. Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli[J]. PROTEIN EXPRESSION AND PURIFICATION,2001,22(2):325-329. |
| APA | Wlad, H,Ballagi, A,Bouakaz, L,Gu, ZY,&Janson, JC.(2001).Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli.PROTEIN EXPRESSION AND PURIFICATION,22(2),325-329. |
| MLA | Wlad, H,et al."Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli".PROTEIN EXPRESSION AND PURIFICATION 22.2(2001):325-329. |
入库方式： OAI收割
来源：过程工程研究所
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