NDLI: Identification of soluble protein fragments by gene fragmentation and genetic selection

Content Provider	PubMed Central
Author	Dyson, Michael R. Perera, Rajika L. Shadbolt, S. Paul Biderman, Lynn Bromek, Krystyna Murzina, Natalia V. Mccafferty, John
Copyright Year	2008
Abstract	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.
Related Links	http://dx.doi.org/10.1093/nar/gkn151
Starting Page	51
File Format	PDF
ISSN	03051048
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	9
Volume Number	36
Language	English
Publisher	Oxford University Press
Publisher Date	2008-03-01
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Genetics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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