The goal of this research is to understand how three different parameters affect single chain variable fragment (scFv) binding capacity. The parameters that were varied include the number of variable complementarity determining regions (CDRs), the number amino acids used to diversify CDRs, and configuration of the structure. How the parameters affect the binding capacity will be tested using the yeast two hybrid assay against five different protein domains. Eight scFv libraries were generated; the genes expressing the scFvs were constructed and the CDRs were randomized using PCR amplification. Genes expressing scFvs were cloned, using the homologous gap repair mechanism in <i>Saccharomyces cerevisiae</I>. Representative members of scFv libraries were sequenced to confirm correct construction.<p>
Library diversity was calculated from the library transformation efficiency. Transformation efficiency refers to the number of cells that grew at the time of transformation of the scFv gene into yeast cells. There were significant differences in the diversity of the scFv libraries, which created difficulty in comparing the library binding capacities. Sequencing the scFv libraries revealed that on average 50% of each library contained correct scFv sequences. The percent of correct sequences within each library was then used to calculate the functional diversity.<p>
The yeast two-hybrid assay was used to screen the scFv libraries for interactions and to test binding capacity. The binding capacity of the scFv libraries was tested and compared in five different yeast two-hybrid assays using five protein domains as the targets for each screen.
The screening results showed that in all cases cyclic scFv libraries had a statistically significant higher binding capacity than linear scFv libraries despite a diversity bias against the cyclic libraries. There was no clear trend in binding capacity with the other two parameters; however, the four amino acid three CDR libraries dominated over the other libraries in almost every screen.<p>
Some of the scFvs isolated from the screens were expressed in <i>E. coli</i> and <i>S. cerevisiae</i>to analyze for proper expression and correct size. All the scFvs that were isolated and analyzed were the correct size and could be purified using a poly histidine tag.<p>
Due to its bioaffinity and specificity, scFvs were constructed to profile disease patterns, and to identify potential drug targets. In addition to its original application to health-related studies, scFvs could also be extended to locate potential metabolic bottlenecks, to alter metabolic flux to enhance productivity, and regulate metabolic bionetworks. Industrial microorganisms are generally carrying more than two sets of chromosomes, making it difficult to be genetically engineered when conventional approaches are employed. With the availability of scFvs as reported in this thesis, we are able to design specific scFvs that selectively bind to target proteins, resulting in re-routing of metabolic flux within the microorganism, toward a high productivity of desired product. ScFvs can be applied to industrial microorganisms directly, leading to the development of new fermentation processes.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-11172008-111943 |
| Date | 19 November 2008 |
| Creators | Bernhard, Wendy Lynn |
| Contributors | Peng, Ding-Yu, Niu, Catherine, Nemati, Mehdi, Luo, Yu, Lin, Yen-Han, Geyer, C. Ronald, Sanche, Steve |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-11172008-111943/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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