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Hot CoFi Blot: A High-Throughput Colony-Based Screen for Identifying More Thermally Stable Protein Variants

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Book cover High-Throughput Protein Production and Purification

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2025))

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Abstract

Highly soluble and stable proteins are desirable for many different applications, from basic science to reaching a cancer patient in the form of a biological drug. For X-ray crystallography—where production of a protein crystal might take weeks and even months—a stable protein sample of high purity and concentration can greatly increase the chances of producing a well-diffracting crystal. For a patient receiving a specific protein drug, its safety, efficacy, and even cost are factors affected by its solubility and stability. Increased protein expression and protein stability can be achieved by randomly altering the coding sequence. As the number of mutants generated might be overwhelming, a powerful protein expression and stability screen is required. In this chapter, we describe a colony filtration technology, which allows us to screen random mutagenesis libraries for increased thermal stability—the Hot CoFi blot. We share how to create the random mutagenesis library, how to perform the Hot CoFi blot, and how to identify more thermally stable clones. We use the Tobacco Etch Virus protease as a target to exemplify the procedure.

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Competing Financial Interests

S.D. and P.N. are co-founders of Evitra AB that owns the commercial rights to this method. I.A. and P.N. are co-founders of DotBio Pte. Ltd., which has licensed this method.

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Authors and Affiliations

  1. DotBio Pte. Ltd. 1 Research Link, #05-30, Singapore, Singapore

    Ignacio Asial & Pär Nordlund

  2. Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden

    Pär Nordlund & Sue-Li Dahlroth

  3. Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden

    Pär Nordlund

Authors
  1. Ignacio Asial
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  2. Pär Nordlund
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  3. Sue-Li Dahlroth
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Corresponding author

Correspondence to Ignacio Asial .

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Editors and Affiliations

  1. Architecture et Fonction des Macromolécules Biologiques (AFMB), Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Marseille cedex 9, France

    Renaud Vincentelli

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Asial, I., Nordlund, P., Dahlroth, SL. (2019). Hot CoFi Blot: A High-Throughput Colony-Based Screen for Identifying More Thermally Stable Protein Variants. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

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