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

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...

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Bibliographic Details
Published in:	Nature communications Vol. 4; no. 1; p. 2901
Main Authors:	Asial, Ignacio, Cheng, Yue Xiang, Engman, Henrik, Dollhopf, Maria, Wu, Binghuang, Nordlund, Pär, Cornvik, Tobias
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 19-12-2013 Nature Publishing Group
Subjects:	38/70 42/40 631/1647/2204 631/61/338 82/47 82/80 Biophysics - methods Cloning, Molecular Crystallography, X-Ray Directed Molecular Evolution Endopeptidases - chemistry Endopeptidases - genetics Gene Library High-Throughput Screening Assays - methods Humanities and Social Sciences Interleukin 1 Receptor Antagonist Protein - chemistry Interleukin 1 Receptor Antagonist Protein - genetics Medicin och hälsovetenskap Models, Molecular multidisciplinary Protein Engineering - methods Protein Stability Science Science (multidisciplinary) Single-Chain Antibodies - chemistry Single-Chain Antibodies - genetics Surface Plasmon Resonance
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Summary:	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. Methods to improve protein stability are important in the biopharmaceutical industry. Here, the authors describe a high-throughput screen to increase protein thermostability and identify thermostable variants from a broad range of proteins.
ISSN:	2041-1723 2041-1723
DOI:	10.1038/ncomms3901