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Colony filtration blotting for screening soluble expression in Escherichia coli

Abstract

We have developed a screen for detecting E. coli colonies that produce soluble recombinant target proteins at the colony level: the colony filtration (CoFi) blot. Colonies are transferred, induced and lysed on a filter membrane that can separate soluble proteins from inclusion bodies. Upon lysis, the soluble proteins diffuse through the filter membrane and are captured on a nitrocellulose membrane. The nitrocellulose membrane is incubated with antibodies or probes specific for the target protein and are then developed. In the resulting image, colonies expressing soluble protein can easily be identified. This protocol can be used to screen thousands of constructs in a matter of days, making it very suitable for expression libraries. The protocol is robust and flexible with regard to lysis conditions, induction temperatures and strains. The method requires only standard laboratory equipment and is based on immunochemicals used for western blotting. The following protocol describes the screening of a DNA library with detection done using chemiluminescence. Depending on induction temperature, the whole procedure can be performed in <2 d.

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Figure 1: Schematic outline of the CoFi procedure from picking up colonies from the master plate on the Durapore membrane to transferring them to the induction plate.
Figure 2: Preparation of filter sandwich and lysis procedure.
Figure 3: The master plate, with regrown colonies, is aligned with the CoFi blot image using the markings.
Figure 4: Three possible outcomes of a CoFi blotting screen of an expression library.

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Acknowledgements

The authors 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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Correspondence to Pär Nordlund.

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P.N. and T.C. are the founders of the company Evitra AB, which may make use of the method described in this article.

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Dahlroth, SL., Nordlund, P. & Cornvik, T. Colony filtration blotting for screening soluble expression in Escherichia coli. Nat Protoc 1, 253–258 (2006). https://doi.org/10.1038/nprot.2006.39

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