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Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli

Nature Methods volume 2, pages 507–509 (2005)Cite this article

Abstract

The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library.

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Figure 1: Illustration and verification of the CoFi blot procedure.
Figure 2: CoFi blot screening of the deletion mutagenesis library of HP16.

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Acknowledgements

We thank V. Lieu for help with purification and sequencing, and B. Engvall for cloning the targets for the benchmarking set. We also thank A. Beuscher, M. Högbom and P. Stenmark for their helpful comments on the manuscript. We would like to acknowledge the Swedish Research Council, the Wallenberg Consortium North, the Göran Gustafsson Foundation for Research in Natural Science and Medicine and the European Community–supported program Structural Proteomics in Europe (SPINE) for financial support.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Stockholm University, S-106 91, Stockholm, Sweden

    Tobias Cornvik, Sue-Li Dahlroth, Audur Magnusdottir, Maria Dolores Herman & Rosemarie Knaust

  2. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden

    Tobias Cornvik, Sue-Li Dahlroth, Audur Magnusdottir, Maria Dolores Herman, Monica Ekberg & Pär Nordlund

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  1. Tobias Cornvik
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  2. Sue-Li Dahlroth
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  3. Audur Magnusdottir
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  4. Maria Dolores Herman
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  6. Monica Ekberg
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Corresponding authors

Correspondence to Monica Ekberg or Pär Nordlund.

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Competing interests

Pär Nordlund and Tobias Cornvik are the founders of the company Evitra AB, which may use the method described in the manuscript

Supplementary information

Supplementary Fig. 1

Verification of the CoFi blot procedure. (PDF 355 kb)

Supplementary Fig. 2

The reproducibility of the CoFi blot method was investigated. (PDF 157 kb)

Supplementary Fig. 3

A schematic representation of the Erase-A-Base process. (PDF 260 kb)

Supplementary Table 1

Proteins used to validate the CoFi-blot method. (PDF 56 kb)

Supplementary Methods (PDF 106 kb)

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Cornvik, T., Dahlroth, SL., Magnusdottir, A. et al. Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli. Nat Methods 2, 507–509 (2005). https://doi.org/10.1038/nmeth767

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