Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli.
Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC 2.4.1.1), types L and H, localized in the plastid and the cytoplasm, respectively. We already isolated and sequenced a cDNA clone encoding the type L isozyme. Presently, a cDNA clone encoding the type H counterpart was isolated from a cDNA library of immature potato tuber by plaque hybridization, using two oligonucleotide probes synthesized based on the partial amino acid sequences of the type H isozyme. The message encodes a polypeptide of 838 amino acid residues. Sequence comparison of the two potato tuber phosphorylase isozymes revealed two major distinctions; the type L isozyme contains a 78-residue insertion in the middle of the polypeptide chain as well as a 50-residue amino-terminal extension. Except for these extra portions, the two isozyme sequences show an identity of 63%. The entire structural gene for the type H isozyme was inserted 3'-downstream of the strong T7 RNA polymerase promoter in the expression plasmid pET-3b. Escherichia coli BL21 (DE3) cells carrying this plasmid produced active phosphorylase upon induction with isopropyl-beta-D-thiogalactoside at 22 degrees C. The expression is entirely dependent on the temperature; the bacteria did not produce a detectable amount of the active enzyme at 37 degrees C. Addition of pyridoxine to the culture medium was effective for the enzyme production.
Institute of Scientific and Industrial Research, Osaka University, Japan.
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Mori
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Tanizawa
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Fukui
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D006005:Phosphorylases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D016415:Sequence Alignment; D011198:Solanum tuberosum / Q000201:enzymology*
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D004247:DNA; D006005:Phosphorylases