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

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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Murine dihydrofolate reductase (mDHFR) can act as a protein solubility reporter. (A)Escherichia coli BL21(DE3) expressing mDHFR fused to tyrosine kinase (1) or SAM domain (2) of the murine EphB2 receptor, c-Fos (3) or GFP (4) were streaked onto minimal agar plates containing ampicillin and IPTG in the presence (+) or absence (−) of trimethoprim. (B) and (C) Growth rate studies of E. coli BL21(DE3) expressing various N-termimal fusion proteins to mDHFR in the absence (B) or presence (C) of trimethoprim. (D) SDS–PAGE western blots for total (T) and soluble (S) expression of EphB2-SAM, GFP, EphB2-TK and KSI expressed as mDHFR fusions. Marker lane (m) = His-tag ladder (Qiagen).
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Figure 1: Murine dihydrofolate reductase (mDHFR) can act as a protein solubility reporter. (A)Escherichia coli BL21(DE3) expressing mDHFR fused to tyrosine kinase (1) or SAM domain (2) of the murine EphB2 receptor, c-Fos (3) or GFP (4) were streaked onto minimal agar plates containing ampicillin and IPTG in the presence (+) or absence (−) of trimethoprim. (B) and (C) Growth rate studies of E. coli BL21(DE3) expressing various N-termimal fusion proteins to mDHFR in the absence (B) or presence (C) of trimethoprim. (D) SDS–PAGE western blots for total (T) and soluble (S) expression of EphB2-SAM, GFP, EphB2-TK and KSI expressed as mDHFR fusions. Marker lane (m) = His-tag ladder (Qiagen).

Mentions: To test if mDHFR could be used as a genetic selection reporter of soluble protein expression, a number of E. coli BL21(DE3) expression clones were streaked onto minimal plates containing ampicillin and IPTG in the presence or absence of TMP. TMP is a potent inhibitor of bacterial DHFR, but does not effectively inhibit murine or human DHFR. Therefore, only bacteria expressing TMP resistant (TMPR) mammalian DHFR will enable growth in the presence of TMP. The strains hosted the expression plasmid pDEST-C102-Dhfr, described previously (1), designed to express cloned open reading frames (ORFs) with a C-terminal mDHFR fusion protein. The clones expressed the tyrosine kinase or SAM domain of the murine EphB2 receptor (EphB2-TK or EphB2-SAM, respectively), the murine transcription factor Fos and Aequorea victoria green fluorescent protein (GFP) mutant (4) engineered for soluble expression in E. coli (12). As shown in Figure 1A, all bacterial strains grew well when streaked onto expression plates in the absence of TMP, but in the presence of TMP only the strains expressing the known soluble proteins EphB2-SAM and GFP (1) grew well (plate segments 2 and 4), whereas the insoluble proteins EphB2-TK and Fos either failed to grow or grew very slowly after a 30 h incubation at 30°C (plate segments 1 and 3, Figure 1). The growth results illustrated in Figure 1A provide evidence that mDHFR can act as a dominant genetic reporter of soluble protein expression. Upstream protein fusions to DHFR that are prone to misfolding and inclusion body formation can perturb the folding and therefore enzymatic activity of DHFR. Additional evidence for the ability of DHFR to act as a solubility reporter came from liquid culture experiments in minimal media containing IPTG and ampicillin, where the growth rates of strains expressing the soluble EphB2-SAM and GFP or insoluble EphB2-TK or ketosteroid isomerase (KSI) were compared in the absence or presence of TMP. As shown in Figure 1B, all expression strains grew equally well in the absence of TMP, but in the presence of TMP (Figure 1C) the strains expressing low or undetectable levels of soluble protein (Figure 1D) displayed markedly reduced growth kinetics.Figure 1.


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)

Murine dihydrofolate reductase (mDHFR) can act as a protein solubility reporter. (A)Escherichia coli BL21(DE3) expressing mDHFR fused to tyrosine kinase (1) or SAM domain (2) of the murine EphB2 receptor, c-Fos (3) or GFP (4) were streaked onto minimal agar plates containing ampicillin and IPTG in the presence (+) or absence (−) of trimethoprim. (B) and (C) Growth rate studies of E. coli BL21(DE3) expressing various N-termimal fusion proteins to mDHFR in the absence (B) or presence (C) of trimethoprim. (D) SDS–PAGE western blots for total (T) and soluble (S) expression of EphB2-SAM, GFP, EphB2-TK and KSI expressed as mDHFR fusions. Marker lane (m) = His-tag ladder (Qiagen).
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: Murine dihydrofolate reductase (mDHFR) can act as a protein solubility reporter. (A)Escherichia coli BL21(DE3) expressing mDHFR fused to tyrosine kinase (1) or SAM domain (2) of the murine EphB2 receptor, c-Fos (3) or GFP (4) were streaked onto minimal agar plates containing ampicillin and IPTG in the presence (+) or absence (−) of trimethoprim. (B) and (C) Growth rate studies of E. coli BL21(DE3) expressing various N-termimal fusion proteins to mDHFR in the absence (B) or presence (C) of trimethoprim. (D) SDS–PAGE western blots for total (T) and soluble (S) expression of EphB2-SAM, GFP, EphB2-TK and KSI expressed as mDHFR fusions. Marker lane (m) = His-tag ladder (Qiagen).
Mentions: To test if mDHFR could be used as a genetic selection reporter of soluble protein expression, a number of E. coli BL21(DE3) expression clones were streaked onto minimal plates containing ampicillin and IPTG in the presence or absence of TMP. TMP is a potent inhibitor of bacterial DHFR, but does not effectively inhibit murine or human DHFR. Therefore, only bacteria expressing TMP resistant (TMPR) mammalian DHFR will enable growth in the presence of TMP. The strains hosted the expression plasmid pDEST-C102-Dhfr, described previously (1), designed to express cloned open reading frames (ORFs) with a C-terminal mDHFR fusion protein. The clones expressed the tyrosine kinase or SAM domain of the murine EphB2 receptor (EphB2-TK or EphB2-SAM, respectively), the murine transcription factor Fos and Aequorea victoria green fluorescent protein (GFP) mutant (4) engineered for soluble expression in E. coli (12). As shown in Figure 1A, all bacterial strains grew well when streaked onto expression plates in the absence of TMP, but in the presence of TMP only the strains expressing the known soluble proteins EphB2-SAM and GFP (1) grew well (plate segments 2 and 4), whereas the insoluble proteins EphB2-TK and Fos either failed to grow or grew very slowly after a 30 h incubation at 30°C (plate segments 1 and 3, Figure 1). The growth results illustrated in Figure 1A provide evidence that mDHFR can act as a dominant genetic reporter of soluble protein expression. Upstream protein fusions to DHFR that are prone to misfolding and inclusion body formation can perturb the folding and therefore enzymatic activity of DHFR. Additional evidence for the ability of DHFR to act as a solubility reporter came from liquid culture experiments in minimal media containing IPTG and ampicillin, where the growth rates of strains expressing the soluble EphB2-SAM and GFP or insoluble EphB2-TK or ketosteroid isomerase (KSI) were compared in the absence or presence of TMP. As shown in Figure 1B, all expression strains grew equally well in the absence of TMP, but in the presence of TMP (Figure 1C) the strains expressing low or undetectable levels of soluble protein (Figure 1D) displayed markedly reduced growth kinetics.Figure 1.

Bottom Line: Inhibition of E. coli dihydrofolate reductase (DHFR) by trimethoprim (TMP) prevents growth, but this can be relieved by murine DHFR (mDHFR).These were found to cluster around the DNA binding ETS domain.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.

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