Production of pokeweed antiviral proteins nontoxic to cells by Mutagenesis of PAP-cDNA |
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Authors: | Yoonkang Hur Ching-Tack Han Sangkyu Park |
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Institution: | (1) Department of Biology, Institute of Biotechnology, Chungnum National Univerisity, 305-764 Taejon, Korea;(2) Department of Life Science, Sogang University, 100-611 Seoul, Korea |
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Abstract: | Pokeweed antiviral protein (PAP), one of ribosome inactivating proteins (RIPs), has very strong toxicity both to prokaryotic
and eukaryotic cells. To produce mutant PAPs nontoxic to cells, the PAP-cDNA was inserted into a yeast-E.coli shuttle vector under the control of galactose promoter, mutagenized using hydroxylamine, and transformed into yeast cells.
Transformed yeast cells were selected on the uracil-deficient plate containing glucose or raffinose, and the yeast cells producing
mutant PAPs nontoxic to cells were then selected on the galactose plate. Eighteen mutants were obtained by immunoblot analyses
of 1,000 transformants: among them, three, ten and five mutants produced unprocessed, mature and truncated PAPs, respectively.
Fourteen PAP mutants among them did not inhibit the yeast cell growth, and showed no or less inhibition of protein synthesisin vitro. Six among fourteen mutants were able to protect TMV infection in coinoculation experiment. The mutant PAPs showing an antiviral
activity either without or reduced RIP activity contain neither the active site mutation nor C-terminal deletion mutation.
These results suggest that both the RIP activity and the antiviral activity will require other amino acid residue(s) besides
the active site and that the antiviral acitivity of PAP can be dissociated from its toxicity. |
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Keywords: | pokeweed antiviral protein ribosome-inactivating protein hydroxylamine mutagenesis deletion mutant antiviral activity |
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