CHLAMYDOMONAS-REINHARDTII THIOREDOXINS - STRUCTURE OF THE GENES-CODING FOR THE CHLOROPLASTIC-M AND CYTOSOLIC-H ISOFORMS - EXPRESSION IN ESCHERICHIA-COLI OF THE RECOMBINANT PROTEINS, PURIFICATION AND BIOCHEMICAL-PROPERTIES

Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for t wo thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acid s (15101 Da) containing a transit peptide of 34 amino acids with a ver y high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11817 Da and no signal sequence. The thioredoxin m gene contains a single intro n and seems to be more archaic in structure than the thioredoxin h gen e, which is split into 4 exons. The cDNA sequences encoding C. reinhar dtii thioredoxins m and h have been integrated into the pET-3d express ion vector, which permits efficient production of proteins in Escheric hia coli cells. A high expression level of recombinant thioredoxins wa s obtained (up to 50 mg/l culture). This has allowed us to study the b iochemical/biophysical properties of the two recombinant proteins. Int erestingly, while the m-type thioredoxin was found to have characteris tics very close to the ones of prokaryotic thioredoxins, the h-type th ioredoxin was quite different with respect to its kinetic behaviour an d, most strikingly, its heat denaturation properties.