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J Microbiol Methods. 2014 May;100:70-6. doi: 10.1016/j.mimet.2014.02.016. Epub 2014 Mar 11.

Heterologous expression of a lectin from Pleurocybella porrigens (PPL) in Phanerochaete sordida YK-624.

Author information

1
Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
2
Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
3
Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
4
Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: ahhirai@ipc.shizuoka.ac.jp.
5
Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: achkawa@ipc.shizuoka.ac.jp.

Abstract

Pleurocybella porrigens is a mushroom-forming fungus, which had been consumed as a traditional food in Japan. However, in 2004, 55 people got poisoned by eating the mushroom and 17 people among them died of acute encephalopathy. We have already reported the purification, characterization, and cDNA cloning of a lectin from the mushroom (PPL) which might have caused the poisoning. Here, we report the heterologous expression of recombinant PPL by basidiomycete Phanerochaete sordida YK-624. The glyceraldehyde 3-phosphate dehydrogenase gene promoter was used to drive the expression of the PPL gene (ppl) in P. sordida YK-624. Furthermore, the signal peptide of lignin peroxidase which is an extracellular protein was used to secrete rPPL into extracellular region. Fifteen regenerated clones were cultured on Kirk HNHC broth, and the presence of lectin activity in the culture broth was checked by agglutination assays. The results indicated that the culture broth of rPPL-6 clone showed the strongest hemagglutination activity, and it was therefore used for subsequent analysis. The heterologous expression of rPPL by P. sordida YK-624 was confirmed by SDS-PAGE, lectin activity by the hemagglutination assay, and mass of rPPL by MALDI-TOF respectively, indicating that the extracellular secretion of rPPL as active form was successful.

KEYWORDS:

Heterologous expression; Lectin; Phanerochaete sordida YK-624; Pleurocybella porrigens

PMID:
24631556
DOI:
10.1016/j.mimet.2014.02.016
[Indexed for MEDLINE]
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