Expression of single chain antibody-encoding genes in fission yeast and tobacco
Davis, George Thomas
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https://hdl.handle.net/2142/23325
Description
Title
Expression of single chain antibody-encoding genes in fission yeast and tobacco
Author(s)
Davis, George Thomas
Issue Date
1992
Doctoral Committee Chair(s)
Jacobs, Thomas W.
Department of Study
Plant Biology
Discipline
Plant Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Genetics
Language
eng
Abstract
The goal of this project was to explore the expression of genes encoding single chain antibodies (SCA) in eukaryotic cells. During the course of this research, a new PCR-based strategy was developed that significantly reduced the time required for SCA gene synthesis. Although initially developed for the synthesis of SCA genes from templates of known sequence, PCR primers have now been developed that have universal applicability for this strategy. It was established that murine codon bias did not preclude efficient expression of SCA genes in E. coli.
Using a SCA that recognized the hapten fluorescein (SCA 4420), it was established that Schizosaccharomyces pombe was a viable SCA expression system. We demonstrated that SCA protein produced by S. pombe was capable of binding hapten in vitro by showing that incubation of the total cell lysate with fluorescein conjugated Sepharose resulted in the selective removal of the SCA protein. The issue of SCA function in vivo was addressed by analyzing transgenic fission yeast by flow cytometry. These analyses were inconclusive; although the SCA 4420 expressing strains displayed the predicted attenuation of fluorescence, so did strains expressing a SCA against glutamine synthetase.
Tobacco transformed with the SCA 4420 encoding gene failed to express the SCA to detectable levels. This is in agreement with recent literature indicating that immunoglobulin or derivatives thereof are not expressed in the plant cell cytoplasm. Whether the impediment to plant cytoplasmic expression occurs at translation or thereafter remains to be determined.
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