Identification of soluble protein fragments by gene fragmentation and genetic selection.
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.
Dyson
Michael R
MR
Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK. md458@cam.ac.uk
Perera
Rajika L
RL
Shadbolt
S Paul
SP
Biderman
Lynn
L
Bromek
Krystyna
K
Murzina
Natalia V
NV
McCafferty
John
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D051379:Mice; D019151:Peptide Library; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D012995:Solubility; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D000911:Antibodies, Monoclonal; D019151:Peptide Library; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D051789:Proto-Oncogene Protein c-fli-1; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase