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Gelonin is a 30 kDa potein toxin from plant Gelonium multiflorum. It is know as one of the ribosome-inactivating protein (RIP) and has rRNA N-glycosidase activity to eukaryote ribosome.

Here, we showed the effect of DnaK Mix on the solubility and activity of the synthesized Gelonin.


CONTENTS

Methods

1. Prepare PUREfrex® 2.0 reaction mixture with and without DnaK Mix.
2. Add template DNA (PCR product of Gelonin).
3. Incubate at 30 or 37 °C for 4 h.
4. Spin to separate the supernatant.

Results

Productivity and Solubility

The amount and solubility of the synthesized Gelonin were confirmed by SDS-PAGE. The solubility was checked by comparing the supernatant with the total reaction mixture. 1 µL reaction volume was applied per lane.

As a result, the amount of Gelonin in the soluble fraction was higher when DnaK Mix was added.  The amount of solubilized Gelonin per reaction solution was higher when synthesized at 37 °C than at 30 °C.




Translation inhibition assay

For the assay, the reaction solution containing the synthesized Gelonin was added to the reaction solution prepared for GFP synthesis, the transcription and translation reactions of GFP were performed, and the fluorescence intensity of the synthesized GFP was compared.

As a result, the fluorescence intensity of GFP became lower when the Gelonin reaction solution was added, indicating that the translation reaction of GFP was inhibited in a Gelonin concentration-dependent manner. The synthesized Gelonin showed higher inhibitory activity when the DnaK Mix was added to the reaction solution at 37 °C rather than 30 °C.


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