Purification and Properties of Arabidopsis thaliana COR (Cold-Regulated) Gene Polypeptides COR15am and COR6.6 Expressed in Escherichia coli

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Abstract
Arabidopsis thaliana cold-regulated genes COR15a and COR6.6 encode 15- and 6.6-kD polypeptides, respectively. The COR15a polypeptide is known to be targeted to chloroplasts and, during import, to be processed to a 9.4-kD polypeptide designated COR15am. The COR6.6 polypeptide is thought to be located in the cytosol. The coding sequences for COR15am and COR6.6 were fused to the bacteriophage T7 promoter and expressed in Escherichia coli. The recombinant polypeptides COR15amr and COR6.6r were purified to near homogeneity using a combination of ammonium sulfate fractionation, ion-exchange chromatography, and adsorption chromatography on hydroxyapatite. COR15amr and the major species of COR15am in chloroplasts co-migrated on both two-dimensional O'Farrell gels and nondenaturing polyacrylamide gels. These data corroborate the site of COR15a processing and indicate no difference in charge or quaternary structure between COR15amr and the major species of COR15am in plants. In contrast, the migration patterns of COR6.6r and COR6.6 on two-dimensional gels suggest that a considerable portion of the COR6.6 population in plants is modified. In the accompanying papers (M.S. Webb, S.J. Gilmour, M.F. Thomashow, P.L. Steponkus [1996] Plant Physiology 111: 301–312; M. Uemura, S.J. Gilmour, M.F. Thomashow, P.L. Steponkus [1996] Plant Physiology 111:313–327), the effects of COR15amr and COR6.6r on the cryostability and lyotropic phase behavior of liposomes are examined.