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B6db references: 1917968

type Journal Article
authors Mori, H.; Tanizawa, K.; Fukui, T.
title Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli
journal J Biol Chem
sel selected
ui 1917968
year (1991)
volume 266
number 28
pages 18446-53
abstract Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC, 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.
last changed 2009/01/12 19:33

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