Purification of CND41; Chloroplast Nucleoid Dna-Binding Protein with Proteolytic Activity

Abstract

CND41 is the major DNA-binding protein of chloroplast nucleoid of photomixotrophically cultured tobacco cells (1). Its cDNA was isolated and DNA sequence was determined (2). The deduced amino acid sequence of CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids. Recombinant CND41 expressed in E. coil showed that lysine-rich region existing in N-terminal of mature protein was necessary for DNA binding. CND41 also has aspartic protease active site and considerable identity against mucoropepsin (about 25 %), an aspartic protease of Mucore miehei (3) over 380 amino acids, while the proteolytic activity has not been determined. The characterization of accumulation of CND41 indicated that there was a negative correlation between CND41 accumulation and chloroplast transcript levels. CND41 was abundant in non- or low-photosynthetic cells as stem and non-selected cultured cells, whereas CND41 level was low in photosynthetic cells like mature leaves and photoautotrophically cultured cells. Transgenic tobacco also indicated that tobacco with less CND41 had higher chloroplast transcripts. From these results, we proposed that CND41 might be a negative regulator for the chloroplast gene expression (2), while the function of DNA binding activity and/or protease activity of CND41 in the regulation was not clear yet. In this study, we purified CND41 and determined its proteolytic activity to characterize CND41 function in vivo.