Isolation and Characterization of Hardening-Induced Proteins in Chlorella vulgaris C-27: Identification of Late Embryogenesis Abundant Proteins

Hardening-induced soluble proteins of Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) were isolated and purified by two-dimensional high-performance liquid chromatography (2D-HPLC) on an anion-exchange column, with subsequent reversed-phase chromatography. Some of the proteins were resolved by SDS-PAGE, characterized by amino-terminal sequencing and identified by searching for homologies in databases. Separation of the soluble proteins during the hardening of Chlorella by a combination of 2D-HPLC and SDS-PAGE revealed that at least 31 proteins were induced or increased in abundance. Of particular interest was the induction after 12 h of a 10-kDa protein with the amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVG and the induction after 6 h of a 14-kDa protein with the amino-terminal sequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminal sequences of these proteins indicated that they were homologous to late embryogenesis abundant (LEA) proteins. Furthermore, the level of a 22-kDa protein also increased after 12 h. The amino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD, indicated that it was homologous to thioredoxin peroxidase.