Novel methods and screens for portein stability engineering

• Main Author: Jose, Asial Ignacio
• Other Authors: Nordlund Paer Lennart
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Biophysics; DRNTU::Science::Biological sciences::Biochemistry; DRNTU::Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/58910
• Institution: Nanyang Technological University

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.