Expression of monovalent fragments derived from a human IgM autoantibody in E. coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity.
A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.
Institute for Medical Immunology, Medical Faculty (Charité), Humboldt University, Berlin, Germany.
S
S
Jahn
D
D
Roggenbuck
B
B
Niemann
E S
ES
Ward
D000595:Amino Acid Sequence; D000918:Antibody Specificity / Q000235:genetics*; D001323:Autoantibodies / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D007075:Immunoglobulin M / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001323:Autoantibodies; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011994:Recombinant Proteins