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KMID : 0380219960290020137
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Journal of Biochemistry and Molecular Biology
1996 Volume.29 No. 2 p.137 ~ p.141
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cDNA Cloning and Overexpression of an Isoperoxidase Gene from Korean-Radish, Raphanus sativus L.
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Park Jong-Hoon
Kim Soung-Soo
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Abstract
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A partial cDNA encoding a Korean radish isoperoxidase was obtained from a cDNA library prepared from 9 day old radish root. In order to obtain Korean radish isoperoxidase cDNA, 5' RACE (rapid amplification cDNA end) PCR was performed and a cDNA (prxK1) encoding a complete structural protein was obtained by RT (reverse transcription)-PCR. Sequence analysis revealed that the length of the cDNA was 945 base pairs, and that of the mRNA transcript was ca. 1.6 kb. The deduced amino acid of the protein were composed of 315 amino acid residues and the protein was 92% homologous to turnip peroxidase, and 46% to 50% homologous to other known peroxidases. The 945 bp cDNA encoding Korean radish isoperoxidase was overexpressed in Escherichia coli up to approximately 9% of total cellular protein. The recombinant fusion protein exhibited 43 kDa on SDS-PAGE analysis and the activity level of the recombinant nonglycosylated protein was two fold higher in IPTG induced cell extracts than that of uninduced ones.
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KEYWORD
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cDNA cloning, 5' RACE (rapid amplification cDNA end) and RT (reverse transcription)-PCR, overexpression
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