Antibody variable domain interface and framework sequence requirements for stability and function by high-throughput experiments
| Autor: | Hung-Ju Hsu, 許鴻儒 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 Protein structural stability and biological function are dictated by the formation of intra-domain cores and inter-domain interfaces. However the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to be elucidated due to the intractability in enumerating all packing possibilities and in assessing the consequences of all the variations. In this work, groups of beta-strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for Sanger sequencing, next generation sequencing and for high-throughput thermal inactivation measurements. The results show that the sequence preferences in the hydrophobic packing cores of the intra-domain residues are strikingly flexible among hydrophobic residues different from the consensus sequence, implying that these residues are coupled indirectly with the antigen-binding through energetic stabilization of the protein structures interpreting by Monte Carlo simulation. By contrast, the inter-domain interface residues are directly coupled with the antigen-binding. The inter-domain interface should be treated as an integral part of the antigen-binding site in antibody engineering for stability. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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