Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine

• Published in: Protein engineering Vol. 6; no. 8; p. 981
• Main Authors: Ward, V K, Schneider, P G, Kreissig, S B, Hammock, B D, Choudary, P V
• Format: Journal Article
• Language: English
• Published: England 01.11.1993
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Atrazine - immunology; Base Sequence; Cloning, Molecular; DNA Primers; Immunoglobulin Fab Fragments - biosynthesis; Immunoglobulin Fab Fragments - genetics; Immunoglobulin gamma-Chains - genetics; Immunoglobulin Heavy Chains - genetics; Immunoglobulin kappa-Chains - genetics; Immunoglobulin Light Chains - genetics; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Proteins - biosynthesis; Recombinant Proteins - immunology; Sequence Analysis, DNA
• ISSN: 0269-2139
• DOI: 10.1093/protein/6.8.981

The Fab region of an IgG2b antibody (AM7B2.1) reactive to the herbicide atrazine was cloned into a plasmid vector using the polymerase chain reaction and two sets of degenerate oligonucleotide primers designed to mimic the amino acid variation at the N-termini of kappa L-chains and gamma H-chains. These primers also provide a secretion signal fused precisely to the antibody gene sequence for secretion of the mature antibody. A further set of universal oligonucleotide primers was developed for the direct sequencing of the VH and CH1 regions of gamma H-chains and the VL and CL regions of kappa L-chains without subcloning and were used to determine the sequence of this antibody. The kappa L-chain was found to not possess a conserved Cys residue at position 23 and the implications of this observation are discussed. The cloned genes were expressed in Escherichia coli using a commercially available T7 RNA polymerase-based plasmid. The clones were also expressed in a T7 RNA polymerase-based system containing an attenuated version of the T7 RNA polymerase promoter, plus a lac promoter placed in an antisense orientation, to enhance plasmid stability. The expressed products were confirmed as atrazine reactive by binding to an atrazine derivative conjugated with alkaline phosphatase.