Abstract
The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1λT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins inE. coli, then purified. The two HIC proteinswere found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1×106 times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.
| Original language | English |
|---|---|
| Pages (from-to) | 1656-1663 |
| Number of pages | 8 |
| Journal | Bioscience, Biotechnology and Biochemistry |
| Volume | 64 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - Jan 1 2000 |
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All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Biotechnology
- Biochemistry
- Applied Microbiology and Biotechnology
- Molecular Biology
- Organic Chemistry
Cite this
Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27. / Honjoh, Ken-ichi; Matsumoto, Hiroko; Shimizu, Hideyuki; Ooyama, Kanae; Tanaka, Kageyuki; Oda, Yuichi; Takata, Ryoji; Joh, Toshio; Suga, Koushirou; Miyamoto, Takahisa; Iio, Masayoshi; Hatano, Shoji.
In: Bioscience, Biotechnology and Biochemistry, Vol. 64, No. 8, 01.01.2000, p. 1656-1663.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27
AU - Honjoh, Ken-ichi
AU - Matsumoto, Hiroko
AU - Shimizu, Hideyuki
AU - Ooyama, Kanae
AU - Tanaka, Kageyuki
AU - Oda, Yuichi
AU - Takata, Ryoji
AU - Joh, Toshio
AU - Suga, Koushirou
AU - Miyamoto, Takahisa
AU - Iio, Masayoshi
AU - Hatano, Shoji
PY - 2000/1/1
Y1 - 2000/1/1
N2 - The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1λT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins inE. coli, then purified. The two HIC proteinswere found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1×106 times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.
AB - The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1λT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins inE. coli, then purified. The two HIC proteinswere found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1×106 times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.
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U2 - 10.1271/bbb.64.1656
DO - 10.1271/bbb.64.1656
M3 - Article
VL - 64
SP - 1656
EP - 1663
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 8
ER -