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dc.contributor.advisorGeyer, Clarence R.en_US
dc.creatorHill, Wayneen_US
dc.date.accessioned2014-06-21T12:00:47Z
dc.date.available2014-06-21T12:00:47Z
dc.date.created2012-11en_US
dc.date.issued2014-06-20en_US
dc.date.submittedNovember 2012en_US
dc.identifier.urihttp://hdl.handle.net/10388/ETD-2012-11-771en_US
dc.description.abstractThe availability of cost-effective high throughput screening assays combined with an enhanced understanding of oncogenesis has driven the development of more potent, specific, and less toxic anti-cancer agents. At the forefront of these advances are immunoglobulin molecules and their fragments. However, difficulties in producing antibodies in sufficient quantity and quality for commercial application have driven the development of alternative systems that can produce antibodies efficiently and cost-effectively. This thesis focuses on the engineering of an antibody fragment referred to as a single chain variable fragment (scFv), which consists of antibody light and heavy chain variable domains fused together by a peptide linker. Although the use of scFvs circumvents many of the issue of full-length antibody production, they still possess their own unique set of difficulties, including stability. In this thesis, we explored the following strategies to increase scFv stability. First, we increased the number of linkers used to join the variable light and heavy domains. We constructed two linear and two cyclic permutated scFvs that contained additional peptide linkers. Two linear permutated scFvs, named Model 1 and Model 3, showed increased stability with calculated melting temperatures (Tms) exceeding that of the unpermutated scFv. The two cyclic scFvs were less stable with Tms less than that of the unpermutated scFv. Second, we mutated light and heavy variable domains by introducing prolines or mutating glycine to alanine in the variable domain framework regions. Sites for proline mutations and glycine to alanine mutations were identified and scFvs containing the mutations were purified and their thermal stability tested. Unfortunately, there were no discernible differences between purified scFv mutants and the control scFv. Third, we designed a new selection/screening strategy using phage display and yeast two-hybrid assays to identify complementarity determining regions on scFvs that increased intracellular stability. We used this strategy to isolate anti-Abl-SH3 scFvs. Transient expression of scFvs in K562 cells indicated that two anti-Abl-SH3 scFv decreased viability.en_US
dc.language.isoengen_US
dc.subjectScFven_US
dc.subjectAntibodiesen_US
dc.subjectPhage Displayen_US
dc.subjectYeast Two-hybrid, Protein Engineeringen_US
dc.titleImproving scFv stability through framework engineeringen_US
thesis.degree.departmentBiochemistryen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M.Sc.)en_US
dc.type.materialtexten_US
dc.type.genreThesisen_US
dc.contributor.committeeMemberMoore, Stanley A.en_US
dc.contributor.committeeMemberLuo, Yuen_US


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