Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27.
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-1lambdaT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1 x 10(6) 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.
Honjoh
K I
KI
Department of Bioscience and Biotechnology, Graduate School, Kyushu University, Fukuoka, Japan. honjoh@agr.kyushu-u.ac.jp
Matsumoto
H
H
Shimizu
H
H
Ooyama
K
K
Tanaka
K
K
Oda
Y
Y
Takata
R
R
Joh
T
T
Suga
K
K
Miyamoto
T
T
Iio
M
M
Hatano
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002708:Chlorella / Q000737:chemistry*; D003451:Cryoprotective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D003451:Cryoprotective Agents; D010940:Plant Proteins; C066246:late embryogenesis abundant protein, plant; D007770:L-Lactate Dehydrogenase