| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1991: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Cucumber ascorbate oxidase is a homodimer glycoprotein of molecular size of about 130 kDa. It is a muticopper enzyme and a subunit contains 4 copper atom in a catalytic center. It is important to elucidate the relation between the structure and the function of the copper enzyme. We have cloned and sequenced a cDNA for cucumber ascorbate oxidase in 1989. At the same time, Huber and coloeagues established the three-dimentional structure analysis of zucchini ascorbate oxidase by X-ray crystallography. They identified the amino acid ligands to Cu atom. The amino acid sequence homology between cucumber and zucchini ascorbate oxidase was 89%. To study, details of the structure and the function, site-directed and random mutations of ascorbate oxidase are a powerful approach. Since an expression of the cucumber gene in microbes is essential in such a study, we have tried recloning of the ascorbate oxidase cDNA in Escherichia coli, Bacillus brevis and Saccharomyces cerevisiae. Although the synt
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hesis of active enzyme in microbes did not succeed, summary of the experiment is shown.
1. Expression of the ascorbate oxidase cDNA in microbial cells.
The ascobate oxidase cDNA was ligated in an E. coli high expression vector, pKK233-2 under tac promoter, and transformed E. coli JMIO5. Gene expression was induced by IPTG in a log phase. Sodium laurylsulfate-polyacrylamide gel electrophoresis and the following Western blotting analysis revealed the formation of a large amount of ascorbate oxidase protein. However, E. coli cells accumulated the protein in an inclusion body and no enzyme activity was detected in several culture conditions including incubation temperature, induction time, medium component, copper concentration so on. Denaturation of the inclusion body by urea or guanidine hydrochloric acid, and the following reconstitution of the ascorbate oxidase protein was not successful. In B. brevis secretion vector system, no ascorbate oxidase mRNA was formed. Ascorbate oxidase protein synthesized in S. cerevisiae cells transformed by the cDNA under PH05 promoter showed no enzyme activity.
2. Cloning of a genomic DNA encoding the ascornate oxidase.
A cDNA has a possibility to have incorrect nucleotides by a miss-reading of reverse transcriptase. A genomic library of cucumber was screened by the ascorbate oxidase cDNA as a probe, and a full-length genomic DNA for the ascorbate oxidase was cloned by gene walking. It contained 4 exons and the nucleotide sequence in the coding region was completely same as that in cDNA. Modification of the expression system in yeast and expression in tobacco cultured cells are under going. Less
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