Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain.
Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 micrograms/ml and 0.5 micrograms/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.
VTT Biotechnical Laboratory, Espoo, Finland.
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Alfthan
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Takkinen
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Sizmann
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Seppälä
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Immonen
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Vanne
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Keränen
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Kaartinen
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Knowles
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Teeri
D000818:Animals; D000915:Antibody Affinity; D002462:Cell Membrane; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D015964:Gene Expression Regulation, Bacterial; D006241:Haptens / Q000276:immunology; D006358:Hot Temperature; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D051379:Mice; D010081:Oxazolone / Q000031:analogs & derivatives / Q000276:immunology; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D004220:Disulfides; D006241:Haptens; D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; C018361:2-phenyloxazolone; D010081:Oxazolone