Novel methods and screens for portein stability engineering
Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. In this thesis, I present different methods developed with the aim of improving protein stability, either by mutagenesis or by interaction...
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| Format: | Theses and Dissertations |
| Language: | English |
| Published: |
2014
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| Online Access: | https://hdl.handle.net/10356/58910 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. In this thesis, I present different methods developed with the aim of improving protein stability, either by mutagenesis or by interaction with a protein partner.For protein stabilization through mutagenesis, a new high-throughput colony-based stability screen is described, which is a direct and biophysical read-out of intrinsic protein stability. As the method is generic and activity independent, it can easily be applied to a wide range of proteins. For protein stabilization through interaction with a protein partner, the use of phage display was explored. As an alternative and complement to phage display, I present the development of a new biophysical screen that monitors protein stabilization upon binding in cells and crude extracts, and its use in de novo binder generation using an autonomous VH domain as scaffold. |
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