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

Author

Listed:
  • 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
    Karolinska Institutet)

  • Tobias Cornvik

    (School of Biological Sciences, Nanyang Technological University)

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.

Suggested Citation

  • Ignacio Asial & Yue Xiang Cheng & Henrik Engman & Maria Dollhopf & Binghuang Wu & Pär Nordlund & Tobias Cornvik, 2013. "Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell," Nature Communications, Nature, vol. 4(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:4:y:2013:i:1:d:10.1038_ncomms3901
    DOI: 10.1038/ncomms3901
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    Cited by:

    1. Yuri Frosi & Yen-Chu Lin & Jiang Shimin & Siti Radhiah Ramlan & Kelly Hew & Alf Henrik Engman & Anil Pillai & Kit Yeung & Yue Xiang Cheng & Tobias Cornvik & Par Nordlund & Megan Goh & Dilraj Lama & Za, 2022. "Engineering an autonomous VH domain to modulate intracellular pathways and to interrogate the eIF4F complex," Nature Communications, Nature, vol. 13(1), pages 1-22, December.
    2. Mary S. Morrison & Tina Wang & Aditya Raguram & Colin Hemez & David R. Liu, 2021. "Disulfide-compatible phage-assisted continuous evolution in the periplasmic space," Nature Communications, Nature, vol. 12(1), pages 1-14, December.

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