Synthesis and expression in Escherichia coli of DNA encoding the murine lambda 1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen.
A 658 bp DNA sequence corresponding to the murine lambda 1 chain of a monoclonal antibody, Se155-4, specific for the Salmonella serotype B O-antigen, was designed using Escherichia coli preferred codons and chemically synthesized by ligation of synthetic fragments into a linearized plasmid followed by transformation into E. coli. A synthetic signal peptide (ompA) was fused to express the L chain as a free polypeptide into the periplasm of E. coli cells. After isolation and purification, heterologous recombination of the E. coli L chain with mouse H chain gave an active antigen-binding protein. The activity was 15-20% when compared to protein created by an equivalent association of isolated natural mouse L and H chains as measured by a direct EIA assay. In inhibition experiments with the polysaccharide antigen, the two proteins showed identical titration curves and 50% inhibition points, indicating comparable KA values.
Anand
N N
NN
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Dubuc
G
G
Mandal
S
S
Phipps
J
J
Gidney
M A
MA
Sinnott
B
B
Young
N M
NM
MacKenzie
C R
CR
Bundle
D R
DR
Narang
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000276:immunology*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004274:DNA, Recombinant / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007146:Immunoglobulin lambda-Chains / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019081:O Antigens; D009844:Oligosaccharides / Q000276:immunology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012475:Salmonella / Q000235:genetics* / Q000276:immunology; D014169:Transformation, Bacterial
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D007146:Immunoglobulin lambda-Chains; D019081:O Antigens; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins