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.
Mori
H
H
Institute of Scientific and Industrial Research, Osaka University, Japan.
Tanizawa
K
K
Fukui
T
T
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