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Identification of soluble protein fragments by gene fragmentation and genetic selection.

Dyson MR, Perera RL, Shadbolt SP, Biderman L, Bromek K, Murzina NV, McCafferty J - Nucleic Acids Res. (2008)

Bottom Line: 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).These were found to cluster around the DNA binding ETS domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK. md458@cam.ac.uk

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.

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Biophysical analysis of selected soluble domains. 1D proton NMR spectra of Fli1 10 (A) fragment and Pecam1 84 fragment. (B) 2D 15N-HSQC spectra results of Fli1 10 (C) fragment and Pecam1 84 fragment (D).
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Figure 6: Biophysical analysis of selected soluble domains. 1D proton NMR spectra of Fli1 10 (A) fragment and Pecam1 84 fragment. (B) 2D 15N-HSQC spectra results of Fli1 10 (C) fragment and Pecam1 84 fragment (D).

Mentions: The proteins were first analysed by 1D 1H NMR. Methyl group proton chemical shift signals in the region of 1.0 to −1.0 p.p.m. disperse when a methyl group is placed in the core of the protein and can be indicative of correct protein folding (20,21). The Fli1 fragment clearly shows some well-dispersed peaks in the region of 1 to −1 p.p.m. (Figure 6A), whereas the Pecam1 fragment does not (Figure 6B). 1H-15N HSQC (Heteronuclear Single Quantum Coherence) NMR spectra (21,22) showed well dispersed and sharp resonances for the Fli1 construct (Figure 6C), but this was not apparent for the Pecam1 cytoplasmic domain (Figure 6D). The most intense and sharp signals in the centre of the Fli1 HSQC spectrum belongs to the disordered His-tag. All of the resonances in the spectrum of Pecam1, a domain of similar size, are at least as sharp, indicating a disordered and well soluble protein. Pecam1 does not form larger aggregates, which would markedly widen the resonances. Taken together, the 1D NMR and 2D NMR data are consistent with the monomeric Fli1 12 construct being folded, whereas the Pecam1 84 fragment is disordered rather than trimeric, as indicated by size exclusion. Disordered proteins are known to occupy a larger volume than compact globular proteins under native conditions.Figure 6.


Identification of soluble protein fragments by gene fragmentation and genetic selection.

Dyson MR, Perera RL, Shadbolt SP, Biderman L, Bromek K, Murzina NV, McCafferty J - Nucleic Acids Res. (2008)

Biophysical analysis of selected soluble domains. 1D proton NMR spectra of Fli1 10 (A) fragment and Pecam1 84 fragment. (B) 2D 15N-HSQC spectra results of Fli1 10 (C) fragment and Pecam1 84 fragment (D).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2396403&req=5

Figure 6: Biophysical analysis of selected soluble domains. 1D proton NMR spectra of Fli1 10 (A) fragment and Pecam1 84 fragment. (B) 2D 15N-HSQC spectra results of Fli1 10 (C) fragment and Pecam1 84 fragment (D).
Mentions: The proteins were first analysed by 1D 1H NMR. Methyl group proton chemical shift signals in the region of 1.0 to −1.0 p.p.m. disperse when a methyl group is placed in the core of the protein and can be indicative of correct protein folding (20,21). The Fli1 fragment clearly shows some well-dispersed peaks in the region of 1 to −1 p.p.m. (Figure 6A), whereas the Pecam1 fragment does not (Figure 6B). 1H-15N HSQC (Heteronuclear Single Quantum Coherence) NMR spectra (21,22) showed well dispersed and sharp resonances for the Fli1 construct (Figure 6C), but this was not apparent for the Pecam1 cytoplasmic domain (Figure 6D). The most intense and sharp signals in the centre of the Fli1 HSQC spectrum belongs to the disordered His-tag. All of the resonances in the spectrum of Pecam1, a domain of similar size, are at least as sharp, indicating a disordered and well soluble protein. Pecam1 does not form larger aggregates, which would markedly widen the resonances. Taken together, the 1D NMR and 2D NMR data are consistent with the monomeric Fli1 12 construct being folded, whereas the Pecam1 84 fragment is disordered rather than trimeric, as indicated by size exclusion. Disordered proteins are known to occupy a larger volume than compact globular proteins under native conditions.Figure 6.

Bottom Line: 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).These were found to cluster around the DNA binding ETS domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, UK. md458@cam.ac.uk

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.

Show MeSH
Related in: MedlinePlus