Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell

Ignacio Asial, Yue Xiang Cheng, Henrik Engman, Maria Dollhopf, Binghuang Wu, Pär Nordlund () and Tobias Cornvik ()
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Ignacio Asial: School of Biological Sciences, Nanyang Technological University
Yue Xiang Cheng: School of Biological Sciences, Nanyang Technological University
Henrik Engman: School of Biological Sciences, Nanyang Technological University
Maria Dollhopf: Karolinska Institutet
Binghuang Wu: School of Biological Sciences, Nanyang Technological University
Pär Nordlund: School of Biological Sciences, Nanyang Technological University
Tobias Cornvik: School of Biological Sciences, Nanyang Technological University

Nature Communications, 2013, vol. 4, issue 1, 1-8

Abstract: Abstract Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. Unfortunately, identifying stabilizing mutations is not trivial since most are neutral or deleterious. Here we describe a high-throughput colony-based stability screen, which is a direct and biophysical read-out of intrinsic protein stability in contrast to traditional indirect activity-based methods. By combining the method with a random mutagenesis procedure, we successfully identify thermostable variants from 10 diverse and challenging proteins, including several biotechnologically important proteins such as a single-chain antibody, a commercial enzyme and an FDA-approved protein drug. We also show that thermostabilization of a protein drug using our approach translates into dramatic improvements in long-term stability. As the method is generic and activity independent, it can easily be applied to a wide range of proteins.

Date: 2013
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DOI: 10.1038/ncomms3901

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