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Novel purification method and antibiotic activity of recombinant Momordica charantia MAP30

View Article: PubMed Central - PubMed

ABSTRACT

Ribosome-inactivating proteins (RIPs) are a group of enzymes originally isolated from plants that possess the ability to damage ribosomes in an irreversible manner, leading to inhibition of protein synthesis in eukaryotic cells. In this study, we aimed to purify recombinant RIPs, investigate their function in the treatment of bacterial infection, and determine their toxicity in mice. We employed a pMAL protein fusion and purification system using E. coli transformed with a plasmid containing MBP-tagged MAP30 cDNA. MBP-tagged MAP30 was purified using a modified novel protocol to effectively produce highly active MAP30 of high purity. In an acute toxicity study in mice, no mortality occurred at doses lower than 1.25 mg/kg. MAP30 at both 0.42 and 0.14 mg/kg induced anti-MAP30 IgG, which reached a maximum titer at week 3. In conclusion, recombinant MAP30 prepared using our purification method possesses bioactivity, and has a synergistic bacteria-killing effect that can significantly reduce the required dosages of chloramphenicol and erythromycin. Therefore, when MAP30 is used in combination with chloramphenicol or erythromycin, it may of benefit in terms of reducing the side effects of the antibiotics, as lower concentrations of antibiotics are required.

No MeSH data available.


RIP activity assay. a Time course of the luciferase activity generated in in vitro translation, measured by luciferin substrate. Various amounts of MAP30 were added as shown. b Concentration-dependent reduction of relative luciferase activity by MAP30. The IC50 value was obtained from the graph
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Fig2: RIP activity assay. a Time course of the luciferase activity generated in in vitro translation, measured by luciferin substrate. Various amounts of MAP30 were added as shown. b Concentration-dependent reduction of relative luciferase activity by MAP30. The IC50 value was obtained from the graph

Mentions: The time course of luciferase activity generated during in vitro translation was examined to measure the activity of MAP30 at different concentrations. Complete inhibition was observed at 100 ng/ml of MAP30 (Fig. 2a). A plot of the luciferase activity values versus MAP30 concentrations at 30 min revealed that the IC50 of MAP30 was 2.29 ng/ml (Fig. 2b). Thus, we established a novel and convenient purification method of MAP30 possessing high RIP activity.Fig. 2


Novel purification method and antibiotic activity of recombinant Momordica charantia MAP30
RIP activity assay. a Time course of the luciferase activity generated in in vitro translation, measured by luciferin substrate. Various amounts of MAP30 were added as shown. b Concentration-dependent reduction of relative luciferase activity by MAP30. The IC50 value was obtained from the graph
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383789&req=5

Fig2: RIP activity assay. a Time course of the luciferase activity generated in in vitro translation, measured by luciferin substrate. Various amounts of MAP30 were added as shown. b Concentration-dependent reduction of relative luciferase activity by MAP30. The IC50 value was obtained from the graph
Mentions: The time course of luciferase activity generated during in vitro translation was examined to measure the activity of MAP30 at different concentrations. Complete inhibition was observed at 100 ng/ml of MAP30 (Fig. 2a). A plot of the luciferase activity values versus MAP30 concentrations at 30 min revealed that the IC50 of MAP30 was 2.29 ng/ml (Fig. 2b). Thus, we established a novel and convenient purification method of MAP30 possessing high RIP activity.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Ribosome-inactivating proteins (RIPs) are a group of enzymes originally isolated from plants that possess the ability to damage ribosomes in an irreversible manner, leading to inhibition of protein synthesis in eukaryotic cells. In this study, we aimed to purify recombinant RIPs, investigate their function in the treatment of bacterial infection, and determine their toxicity in mice. We employed a pMAL protein fusion and purification system using E. coli transformed with a plasmid containing MBP-tagged MAP30 cDNA. MBP-tagged MAP30 was purified using a modified novel protocol to effectively produce highly active MAP30 of high purity. In an acute toxicity study in mice, no mortality occurred at doses lower than 1.25 mg/kg. MAP30 at both 0.42 and 0.14 mg/kg induced anti-MAP30 IgG, which reached a maximum titer at week 3. In conclusion, recombinant MAP30 prepared using our purification method possesses bioactivity, and has a synergistic bacteria-killing effect that can significantly reduce the required dosages of chloramphenicol and erythromycin. Therefore, when MAP30 is used in combination with chloramphenicol or erythromycin, it may of benefit in terms of reducing the side effects of the antibiotics, as lower concentrations of antibiotics are required.

No MeSH data available.