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A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.

Tikhonova EB, Ethayathulla AS, Su Y, Hariharan P, Xie S, Guan L - Sci Rep (2015)

Bottom Line: Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein.All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins.Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.

ABSTRACT
A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

No MeSH data available.


Related in: MedlinePlus

Isolation of a transcription blocker by an in vivo functional screen.(a), Illustration of the melibiose metabolic pathway in E. coli. For all the following panels, E. coli Tuner cells harboring pCS19/ET (vector control), pCS19/ANK-N5C-62 (protein control), or pCS19/ANK-N5C-281, respectively, were used. The plasmids are described in Table 1. Ampicillin at 100 μg/mL, melibiose at 30 mM, and IPTG at 0.3 mM were used unless otherwise described. (b), Protein-concentration dependent effect. The cells carrying a given plasmid were tested for melibiose and glucose fermentation, respectively, on MacConkey agar plates with ampicillin, sugar, and different concentrations of IPTG. The expression of ANK-N5C proteins was analyzed with His-tag antibody by Western blotting using the cells collected from above melibiose-containing plates. (c), Cell growth on M9 minimal media supplemented with ampicillin, IPTG, and 10 mM melibiose. (d), Detection of MelA and MelB activity and expression. Cells were grown at 30°C in LB media containing 0.5% glycerol, ampicillin, and IPTG with or without 10 mM melibiose, and used for activity and expression of MelA (top two panels) and MelB (bottom two panels). The activity of MelA and MelB (bars) are expressed as α-NPG hydrolysis and uptake of [1-3H]melibiose (0.4 mM, 10 mCi/mmol), respectively. Expression of MelA and MelB proteins (images) were analyzed by Western blot. A total of 40 μg cell extracts or membrane proteins, as well as 100 ng of purified MelA or MelB were loaded on SDS-12% PAGE. Error bars, S.E.M.; n = 3-5. (e), RT-PCR. Total RNA was purified from cells grown described in panel d. The reverse transcriptase enzyme mix was treated for 10 min at 95°C before PCR reaction for control. Products were analyzed on 3% agarose gel. Water, instead of cells, was used for control.
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f3: Isolation of a transcription blocker by an in vivo functional screen.(a), Illustration of the melibiose metabolic pathway in E. coli. For all the following panels, E. coli Tuner cells harboring pCS19/ET (vector control), pCS19/ANK-N5C-62 (protein control), or pCS19/ANK-N5C-281, respectively, were used. The plasmids are described in Table 1. Ampicillin at 100 μg/mL, melibiose at 30 mM, and IPTG at 0.3 mM were used unless otherwise described. (b), Protein-concentration dependent effect. The cells carrying a given plasmid were tested for melibiose and glucose fermentation, respectively, on MacConkey agar plates with ampicillin, sugar, and different concentrations of IPTG. The expression of ANK-N5C proteins was analyzed with His-tag antibody by Western blotting using the cells collected from above melibiose-containing plates. (c), Cell growth on M9 minimal media supplemented with ampicillin, IPTG, and 10 mM melibiose. (d), Detection of MelA and MelB activity and expression. Cells were grown at 30°C in LB media containing 0.5% glycerol, ampicillin, and IPTG with or without 10 mM melibiose, and used for activity and expression of MelA (top two panels) and MelB (bottom two panels). The activity of MelA and MelB (bars) are expressed as α-NPG hydrolysis and uptake of [1-3H]melibiose (0.4 mM, 10 mCi/mmol), respectively. Expression of MelA and MelB proteins (images) were analyzed by Western blot. A total of 40 μg cell extracts or membrane proteins, as well as 100 ng of purified MelA or MelB were loaded on SDS-12% PAGE. Error bars, S.E.M.; n = 3-5. (e), RT-PCR. Total RNA was purified from cells grown described in panel d. The reverse transcriptase enzyme mix was treated for 10 min at 95°C before PCR reaction for control. Products were analyzed on 3% agarose gel. Water, instead of cells, was used for control.

Mentions: In E. coli, the mel operon (Fig. 3a), which encodes MelA and MelB, is needed for melibiose metabolism2930. For a pilot study, we developed a colony-based functional screening method to identify ANK-N5C proteins inhibiting melibiose fermentation as described in Methods (Fig. 3a). By expressing an ANK-N5C protein encoded by a pCS19/FX-derived plasmid (Table 1) in the Tuner cell (lacY−) on melibiose-containing MacConkey agar plates, we identified one yellow colony and 35 other colonies with reduced color from approximately 5 × 105 colonies (Fig. S1a–c). Some clones affect cell growth and some affect glucose fermentation. In this study, we only focus on the one that completely inhibits melibiose fermentation.


A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.

Tikhonova EB, Ethayathulla AS, Su Y, Hariharan P, Xie S, Guan L - Sci Rep (2015)

Isolation of a transcription blocker by an in vivo functional screen.(a), Illustration of the melibiose metabolic pathway in E. coli. For all the following panels, E. coli Tuner cells harboring pCS19/ET (vector control), pCS19/ANK-N5C-62 (protein control), or pCS19/ANK-N5C-281, respectively, were used. The plasmids are described in Table 1. Ampicillin at 100 μg/mL, melibiose at 30 mM, and IPTG at 0.3 mM were used unless otherwise described. (b), Protein-concentration dependent effect. The cells carrying a given plasmid were tested for melibiose and glucose fermentation, respectively, on MacConkey agar plates with ampicillin, sugar, and different concentrations of IPTG. The expression of ANK-N5C proteins was analyzed with His-tag antibody by Western blotting using the cells collected from above melibiose-containing plates. (c), Cell growth on M9 minimal media supplemented with ampicillin, IPTG, and 10 mM melibiose. (d), Detection of MelA and MelB activity and expression. Cells were grown at 30°C in LB media containing 0.5% glycerol, ampicillin, and IPTG with or without 10 mM melibiose, and used for activity and expression of MelA (top two panels) and MelB (bottom two panels). The activity of MelA and MelB (bars) are expressed as α-NPG hydrolysis and uptake of [1-3H]melibiose (0.4 mM, 10 mCi/mmol), respectively. Expression of MelA and MelB proteins (images) were analyzed by Western blot. A total of 40 μg cell extracts or membrane proteins, as well as 100 ng of purified MelA or MelB were loaded on SDS-12% PAGE. Error bars, S.E.M.; n = 3-5. (e), RT-PCR. Total RNA was purified from cells grown described in panel d. The reverse transcriptase enzyme mix was treated for 10 min at 95°C before PCR reaction for control. Products were analyzed on 3% agarose gel. Water, instead of cells, was used for control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4308713&req=5

f3: Isolation of a transcription blocker by an in vivo functional screen.(a), Illustration of the melibiose metabolic pathway in E. coli. For all the following panels, E. coli Tuner cells harboring pCS19/ET (vector control), pCS19/ANK-N5C-62 (protein control), or pCS19/ANK-N5C-281, respectively, were used. The plasmids are described in Table 1. Ampicillin at 100 μg/mL, melibiose at 30 mM, and IPTG at 0.3 mM were used unless otherwise described. (b), Protein-concentration dependent effect. The cells carrying a given plasmid were tested for melibiose and glucose fermentation, respectively, on MacConkey agar plates with ampicillin, sugar, and different concentrations of IPTG. The expression of ANK-N5C proteins was analyzed with His-tag antibody by Western blotting using the cells collected from above melibiose-containing plates. (c), Cell growth on M9 minimal media supplemented with ampicillin, IPTG, and 10 mM melibiose. (d), Detection of MelA and MelB activity and expression. Cells were grown at 30°C in LB media containing 0.5% glycerol, ampicillin, and IPTG with or without 10 mM melibiose, and used for activity and expression of MelA (top two panels) and MelB (bottom two panels). The activity of MelA and MelB (bars) are expressed as α-NPG hydrolysis and uptake of [1-3H]melibiose (0.4 mM, 10 mCi/mmol), respectively. Expression of MelA and MelB proteins (images) were analyzed by Western blot. A total of 40 μg cell extracts or membrane proteins, as well as 100 ng of purified MelA or MelB were loaded on SDS-12% PAGE. Error bars, S.E.M.; n = 3-5. (e), RT-PCR. Total RNA was purified from cells grown described in panel d. The reverse transcriptase enzyme mix was treated for 10 min at 95°C before PCR reaction for control. Products were analyzed on 3% agarose gel. Water, instead of cells, was used for control.
Mentions: In E. coli, the mel operon (Fig. 3a), which encodes MelA and MelB, is needed for melibiose metabolism2930. For a pilot study, we developed a colony-based functional screening method to identify ANK-N5C proteins inhibiting melibiose fermentation as described in Methods (Fig. 3a). By expressing an ANK-N5C protein encoded by a pCS19/FX-derived plasmid (Table 1) in the Tuner cell (lacY−) on melibiose-containing MacConkey agar plates, we identified one yellow colony and 35 other colonies with reduced color from approximately 5 × 105 colonies (Fig. S1a–c). Some clones affect cell growth and some affect glucose fermentation. In this study, we only focus on the one that completely inhibits melibiose fermentation.

Bottom Line: Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein.All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins.Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology &Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.

ABSTRACT
A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

No MeSH data available.


Related in: MedlinePlus