Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine.
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.
Ward
V K
VK
UC Davis Antibody Engineering Laboratory.
Schneider
P G
PG
Kreissig
S B
SB
Hammock
B D
BD
Choudary
P V
PV
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D001280:Atrazine / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007142:Immunoglobulin gamma-Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007142:Immunoglobulin gamma-Chains; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; D001280:Atrazine