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Recognition of E. coli Tryptophan Synthase by Single-Chain Fv Fragments: Comparison of PCR-Cloning Variants with the Parental Antibodies

Abstract : The use of a recombinant antibody fragment instead of a complete antibody, as a conformational probe for protein structure and folding studies, can be technically advantageous provided that the recombinant fragment and its parental antibody recognize the antigen through the same mechanism. Monoclonal antibodies mAb19 and mAb93 are directed against the TrpB 2 subunit of Escherichia coli tryptophan synthase and they have been extensively used as conformational probes of this protein. DNA sequences coding for single-chain variable fragments (scFv) of mAb19 and mAb93 were cloned and assembled by reverse transcription of the mRNAs from hybridomas and PCR amplification. A specialized plasmid vector, pFBX, was constructed; it enabled to express the scFvs as hybrids with the maltose-binding protein (MalE) in E. coli, and to purify them by affinity chromatography on cross-linked amylose. Six independent clones were sequenced for each hybridoma. All of them had differences in their nucleotide and amino acid sequences. A competition ELISA and the BIAcore TM biosensor apparatus were used to compare the energetics and kinetics with which the parental antibodies and the hybrids bound TrpB 2. The antigen binding properties of the hybrids were close to those of the parental antibodies and they were only weakly affected by the differences of sequence between the clones, with one exception. The stability of one of the hybrids and its antigen binding properties were strongly modified by a change of Gln6 into Glu, introduced into its V H domain by the PCR primers. Simple models of bimolecular interaction did not fully account for the kinetic profiles obtained with the parental antibodies and the hybrids, and this complexity suggested the existence of a conformational heterogeneity in these molecules.
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Submitted on : Friday, January 14, 2022 - 6:48:40 AM
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François Brégégère, Patrick England, Lisa Djavadi-Ohaniance, Hugues Bedouelle. Recognition of E. coli Tryptophan Synthase by Single-Chain Fv Fragments: Comparison of PCR-Cloning Variants with the Parental Antibodies. Journal of Molecular Recognition, Wiley, 1997, 10 (4), pp.169-181. ⟨10.1002/(SICI)1099-1352(199707/08)10:4<169::AID-JMR362>3.0.CO;2-U⟩. ⟨pasteur-03525674⟩



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