Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli
UMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyDocument Type
Journal ArticlePublication Date
2001-07-05Keywords
Amino Acid SequenceBinding Sites, Antibody
Capsid
*Capsid Proteins
Chromatography, Agarose
Drug Stability
Escherichia coli
Fermentation
Immunoglobulin Fab Fragments
purification
Molecular Sequence Data
Recombinant Proteins
Sepharose
Tobacco Mosaic Virus
Biochemistry, Biophysics, and Structural Biology
Pharmacology, Toxicology and Environmental Health
Metadata
Show full item recordAbstract
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 fermentors. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degrees C), (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 fermentors. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the crude 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 NH(2)-CGS YNR GSF SQS SGLV-CONH(2) had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%.Source
Protein Expr Purif. 2001 Jul;22(2):325-9. Link to article on publisher's siteDOI
10.1006/prep.2001.1444Permanent Link to this Item
http://hdl.handle.net/20.500.14038/25991PubMed ID
11437609Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1006/prep.2001.1444