Construction and mutagenesis of an anti-insulin single chain Fv (scFv) using protein engineering techniques: Identification of peptide mimotopes which inhibit scFv-insulin binding.

Persistent Link:
http://hdl.handle.net/10150/186310
Title:
Construction and mutagenesis of an anti-insulin single chain Fv (scFv) using protein engineering techniques: Identification of peptide mimotopes which inhibit scFv-insulin binding.
Author:
Lake, Douglas Fletcher.
Issue Date:
1993
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Peptide mimotopes were found using random peptide library technology (Selectide technology) which bind to the paratope of an anti-insulin monoclonal antibody. These peptides inhibit insulin from binding to the anti-insulin antibody, indicating that they share at least part of the paratope for insulin. The 50% inhibitory concentrations (IC50) of certain peptides were determined to be less than one order of magnitude from that of insulin. The immunoglobulin variable region genes of the anti-insulin antibody were rescued and cloned independently as V(H) and V(k). A single chain Fv (scFv) was constructed using a polymerase chain reaction (PCR) assembly technique from the anti-insulin V(H) and V(k). The scFv demonstrated similar, but not identical binding properties to the parental anti-insulin antibody. Binding of four of six peptide mimotopes was localized to the variable region of the anti-insulin heavy chain. The heavy chain alone was also able to bind insulin. None of the peptides bound to the variable region of the kappa light chain. Mutagenesis studies indicated that complementarity determining region (CDR) 1 and CDR3 of the heavy chain were important for binding to insulin and to the peptides. Position 99 in CDR3 of the heavy chain was found to be most critical for binding to insulin. Mutational analysis of position 99 revealed that glycine and alanine are acceptable residues for position 99, whereas valine and threonine are not. This finding suggested that residue size, not hydrophobicity determined whether the antibody could bind to insulin.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Dissertations, Academic.; Molecular biology.; Immunology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Microbiology and Immunology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Hersh, Evan M.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleConstruction and mutagenesis of an anti-insulin single chain Fv (scFv) using protein engineering techniques: Identification of peptide mimotopes which inhibit scFv-insulin binding.en_US
dc.creatorLake, Douglas Fletcher.en_US
dc.contributor.authorLake, Douglas Fletcher.en_US
dc.date.issued1993en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractPeptide mimotopes were found using random peptide library technology (Selectide technology) which bind to the paratope of an anti-insulin monoclonal antibody. These peptides inhibit insulin from binding to the anti-insulin antibody, indicating that they share at least part of the paratope for insulin. The 50% inhibitory concentrations (IC50) of certain peptides were determined to be less than one order of magnitude from that of insulin. The immunoglobulin variable region genes of the anti-insulin antibody were rescued and cloned independently as V(H) and V(k). A single chain Fv (scFv) was constructed using a polymerase chain reaction (PCR) assembly technique from the anti-insulin V(H) and V(k). The scFv demonstrated similar, but not identical binding properties to the parental anti-insulin antibody. Binding of four of six peptide mimotopes was localized to the variable region of the anti-insulin heavy chain. The heavy chain alone was also able to bind insulin. None of the peptides bound to the variable region of the kappa light chain. Mutagenesis studies indicated that complementarity determining region (CDR) 1 and CDR3 of the heavy chain were important for binding to insulin and to the peptides. Position 99 in CDR3 of the heavy chain was found to be most critical for binding to insulin. Mutational analysis of position 99 revealed that glycine and alanine are acceptable residues for position 99, whereas valine and threonine are not. This finding suggested that residue size, not hydrophobicity determined whether the antibody could bind to insulin.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectDissertations, Academic.en_US
dc.subjectMolecular biology.en_US
dc.subjectImmunology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairHersh, Evan M.en_US
dc.contributor.committeememberHendrix, Mary J.C.en_US
dc.identifier.proquest9333316en_US
dc.identifier.oclc717653028en_US
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