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Nucleic Acids Research Advance Access originally published online on April 17, 2008
Nucleic Acids Research 2008 36(9):e51; doi:10.1093/nar/gkn151
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Nucleic Acids Research, 2008, Vol. 36, No. 9 e51
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Identification of soluble protein fragments by gene fragmentation and genetic selection

Michael R. Dyson*, Rajika L. Perera, S. Paul Shadbolt, Lynn Biderman, Krystyna Bromek, Natalia V. Murzina and John McCafferty

Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK

*To whom correspondence should be addressed. Tel: +44 1223 339321; Fax: +44 1223 333345; Email: md458{at}cam.ac.uk

Received December 23, 2007. Revised March 16, 2008. Accepted March 17, 2008.

We describe a new method, which identifies protein fragments for soluble expression in Escherichia coli from a randomly fragmented gene library. Inhibition of E. coli dihydrofolate reductase (DHFR) by trimethoprim (TMP) prevents growth, but this can be relieved by murine DHFR (mDHFR). Bacterial strains expressing mDHFR fusions with the soluble proteins green fluroscent protein (GFP) or EphB2 (SAM domain) displayed markedly increased growth rates with TMP compared to strains expressing insoluble EphB2 (TK domain) or ketosteroid isomerase (KSI). Therefore, mDHFR is affected by the solubility of fusion partners and can act as a reporter of soluble protein expression. Random fragment libraries of the transcription factor Fli1 were generated by deoxyuridine incorporation and endonuclease V cleavage. The fragments were cloned upstream of mDHFR and TMP resistant clones expressing soluble protein were identified. These were found to cluster around the DNA binding ETS domain. A selected Fli1 fragment was expressed independently of mDHFR and was judged to be correctly folded by various biophysical methods including NMR. Soluble fragments of the cell-surface receptor Pecam1 were also identified. This genetic selection method was shown to generate expression clones useful for both structural studies and antibody generation and does not require a priori knowledge of domain architecture.

Present addresses: Rajika L. Perera, GlaxoSmithKline Research and Development, New Frontiers Science Park, Harlow, Essex, CM19 5AD, UK S. Paul Shadbolt, Lonza Biologics PLC, 228 Bath Road, Slough, Berkshire, SL1 4DN, UK Lynn Biderman, Department of Biological Sciences, Columbia University, New York, New York 10027, USA


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