Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli.
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%.
Wlad
H
H
Center for Surface Biotechnology, Uppsala Biomedical Center, Uppsala, SE-751 23, Sweden.
Ballagi
A
A
Bouakaz
L
L
Gu
Z
Z
Janson
J C
JC
D000595:Amino Acid Sequence; D001666:Binding Sites, Antibody; D002213:Capsid / Q000276:immunology*; D036022:Capsid Proteins; D002847:Chromatography, Agarose; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000276:immunology*; D005285:Fermentation; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives*; D014027:Tobacco Mosaic Virus / Q000276:immunology
D036022:Capsid Proteins; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; C090158:cp protein, tobacco vein-banding mosaic virus; C047264:sepharose CL 6B; D012685:Sepharose