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

Vernon K. Ward, Peter G. Sehneider, Sabine B. Kreissig, Bruce D. Hammock, Prabhakara V Choudary

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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 χL-chains and TH-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 Cm regions of γB-chains and the VL and CL regions of χL-chains without subcloning and were used to determine the sequence of this antibody. The χL-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 17 RNA polymerasebased 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.

Original languageEnglish (US)
Pages (from-to)981-988
Number of pages8
JournalProtein Engineering, Design and Selection
Volume6
Issue number8
DOIs
StatePublished - Nov 1993

Fingerprint

Atrazine
Immunoglobulin Fab Fragments
Monoclonal antibodies
Cloning
Herbicides
Antibodies
Organism Cloning
RNA
Monoclonal Antibodies
Plasmids
DNA Primers
Oligonucleotides
Genes
Polymerase chain reaction
Phosphatases
Escherichia coli
Alkaline Phosphatase
Amino acids
Clone Cells
Derivatives

Keywords

  • Antibody cloning
  • Antibody expression
  • Atrazine
  • Pesticide analysis
  • Recombinant antiboby

ASJC Scopus subject areas

  • Pharmacology
  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Molecular Biology
  • Bioengineering
  • Biotechnology
  • Biochemistry

Cite this

Cloning, sequencing and expression of the fab fragment of a monoclonal antibody to the herbicide atrazine. / Ward, Vernon K.; Sehneider, Peter G.; Kreissig, Sabine B.; Hammock, Bruce D.; Choudary, Prabhakara V.

In: Protein Engineering, Design and Selection, Vol. 6, No. 8, 11.1993, p. 981-988.

Research output: Contribution to journalArticle

Ward, Vernon K. ; Sehneider, Peter G. ; Kreissig, Sabine B. ; Hammock, Bruce D. ; Choudary, Prabhakara V. / Cloning, sequencing and expression of the fab fragment of a monoclonal antibody to the herbicide atrazine. In: Protein Engineering, Design and Selection. 1993 ; Vol. 6, No. 8. pp. 981-988.
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