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Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli
Alternative TitleProtein Expr. Purif.
Wlad, H; Ballagi, A; Bouakaz, L; Gu, ZY; Janson, JC
2001-07-01
Source PublicationPROTEIN EXPRESSION AND PURIFICATION
ISSN1046-5928
Volume22Issue:2Pages:325-329
AbstractWe 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.; 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.
KeywordMass-transport Limitation Biosensor Technology Bacterial Expression Interaction Kinetics Chain Antibodies Proteins
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
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Indexed BySCI
Language英语
WOS KeywordMASS-TRANSPORT LIMITATION ; BIOSENSOR TECHNOLOGY ; BACTERIAL EXPRESSION ; INTERACTION KINETICS ; CHAIN ; ANTIBODIES ; PROTEINS
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS SubjectBiochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS IDWOS:000169703900020
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Cited Times:16[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/5782
Collection研究所(批量导入)
Affiliation1.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
Recommended Citation
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.
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