Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions.
In this report, we describe the expression system that enabled us to produce in Escherichia coli the Fab fragment of a mouse IgM that has previously been shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. In our system, both light chain and heavy chain fragments were put under the control of the malE promoter. The light chain was fused to the MalE signal sequence, while the heavy chain variable and first constant region were fused to the alkaline phosphatase signal sequence. In this system, after induction of the promoter with maltose, the Fab fragment could be detected in a periplasmic extract of the bacteria by Western blotting and also by ELISA. This Fab fragment was purified on a goat anti-mouse immunoglobulin immunoadsorbent and biotinylated. The Fab fragment produced by E.coli reacted with the trinitrophenyl (TNP) hapten and F(ab')2 fragments of mouse IgG and these reactivities could be specifically inhibited by the corresponding soluble antigens. The dissociation constants of this Fab were 1.65 x 10(-6) M for TNP and 5 x 10(-6) M for IgG F(ab')2 fragments, indicating that the affinity of the Fab fragment compared with that of the whole IgM molecule was similar for TNP but was lower for IgG F(ab')2 fragments.
Adib-Conquy
M
M
CNRS URA 359, Institut Pasteur, Paris, France.
Gilbert
M
M
Christodoulou
C
C
Avrameas
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins