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-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.