A monoclonal antibody generated against a recombinant peptide fragment of the B3 domain of carcinoembryonic antigen reacts with intact carcinoembryonic antigen.
The chemical synthesis of a gene coding for a polypeptide of 77 amino acid residues (designated ceaB3) representing a fragment of the CEA-B3 domain of carcinoembryonic antigen (CEA) was achieved. The ceaB3 fragment was cloned into the plasmid pLZPWB1 at the C-terminus of a derivative lacZMF of the lacZ gene, devoid of methionine and cysteine amino acid residues. The fusion protein lacZMF-ceaB3 represented approx. 30% of total proteins expressed after induction. The fusion protein was formed as inclusion bodies. Simple washing steps led to an insoluble fusion protein which was of approx. 80% purity. Another fusion gene was generated by inserting ceaB3 between the malE gene encoding maltose binding protein (mbp) and lacZ alpha of the pmal-c2 vector. Expression of the resulting pmal-c2-ceaB3-lacZ alpha yielded the fusion protein mbp-ceaB3-lacZ alpha with a molecular mass of 57.94 kDa, which was obtained as a soluble protein in almost homogeneous form after affinity chromatography employing amylopectin. Polyclonal sheep anti-CEA antiserum specifically reacted with fusion proteins lacZMF-ceaB3 and mbp-ceaB3-lacZ alpha. A monoclonal antibody CEA/HK2 was generated employing lacZMF-ceaB3 for immunization and CEA for screening purposes. The mAB CEA/HK2 specifically recognized CEA in immunoblots. The described experimental strategy should be generally applicable for generation of fusion proteins. These fusion proteins are suitable for epitope characterization of existing antibodies, production of regiospecific polyclonal or monoclonal antibodies.
Hagendorff
G
G
Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.
Hanes
J
J
von der Kammer
H
H
Scheit
K H
KH
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000737:chemistry / Q000276:immunology*; D002352:Carrier Proteins / Q000235:genetics; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005260:Female; D058619:Maltose-Binding Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000276:immunology*; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D004247:DNA; D001616:beta-Galactosidase