Development of high throughput screening systems for the efficient production of antibody fragments in Escherichia coli

Recombinant antibodies and antibody fragments have become powerful tools for therapy, in vivo and in vitro diagnostics, and laboratory research. However, the production of antibody fragments in high yield for preclinical and clinical trials can be a serious bottleneck in drug discovery. This dissertation describes the development of novel screening systems for isolating antibody fragments and alternatively, E. coli genes, that facilitate expression in E. coli. In the first part of this work, we have developed a screening system for isolating Fab mutants exhibiting 4~5 fold higher expression level at 37oC compared to the parental Fab, by utilizing the APEx 2-hybrid system and multi-color FACS as a screening tool. In the APEx 2-hybrid system, the bacterial periplasm constitutes the milieu for the association of membrane-anchored bait protein and solubly expressed, epitope-tagged prey protein. Upon disruption of the outer membrane, only prey proteins that bind to the bait remain cell-associated and are detected by flow cytometry using fluorescently labeled anti-epitope antibodies. In the second part of this work we developed a new strategy to engineer scFv that can be expressed in soluble and active form in the absence of disulfide bonds. This was achieved using a strain incapable of forming disulfide bonds in proteins expressed in its periplasm in combination with the APEx 2-hybrid system. The selected clones exhibited higher solubility, activity, and stability than that of the wild type scFv in the reducing condition of the cytoplasm. Finally, we sought to isolate E. coli gene fragments that can enhance IgG production in the periplasm of E. coli by a newly developed screening system based on soluble expression of IgG and E. coli genomic fragments. The isolated gene fragments, which are located between moeA and iaaA in the E. coli chromosome, improved the total expression of polypeptides of IgG and also assembly of IgG.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

The University of Texas at Austin

Year dc:date.issued

2008

Author dc:creator

• Seo, Min Jeong, 1979-

Advisor dc:contributor.advisor

• Georgiou, George

Statement dc:rights

• Copyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/2152/17756

OAI identifier oai:identifier

oai:repositories.lib.utexas.edu:2152/17756