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Nat Commun. 2013;4:2901. doi: 10.1038/ncomms3901.

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

Author information

1
School of Biological Sciences, Nanyang Technological University, 639798 Singapore, Singapore.
2
Division of Biophysics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-171 11 Stockholm, Sweden.
3
1] School of Biological Sciences, Nanyang Technological University, 639798 Singapore, Singapore [2] Division of Biophysics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-171 11 Stockholm, Sweden.

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.

PMID:
24352381
DOI:
10.1038/ncomms3901
[Indexed for MEDLINE]

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