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 |
| |
Authors: | Mariana Stein Jean-Pierre Jacquot Emmanuelle Jeannette Paulette Decottignies Michael Hodges Jean-Marc Lancelin Virginie Mittard Jean-Marie Schmitter Myroslawa Miginiac-Maslow |
| |
Affiliation: | (1) Laboratoire de Physiologie Végétale Moléculaire, URA 1128 CNRS, Université de Paris-Sud, Bâtiment 630, 91405 Orsay Cedex, France;(2) Institut de Biologie Structurale, CEA-CNRS 41, Avenue des Martyrs 38027, Grenoble Cedex, France;(3) Laboratoire de Biochimie Ecole Polytechnique, 91128 Palaiseau Cedex, France |
| |
Abstract: | Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15 101 Da) containing a transit peptide of 34 amino acids with a very 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 intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.Abbreviations DTT dithiothreitol - FBPase Fructose 1,6-biphosphate phosphatase - FTR ferredoxin-thioredoxin reductase - IPTG isopropyl thiogalactoside - NADP-MDH NADPH-dependent malate dehydrogenase - NMR nuclear magnetic resonance - NTR NADPH-dependent thioredoxin reductaseDedicated to the memory of Claude Crétin |
| |
Keywords: | thioredoxin disulfide reduction light regulation Chlamydomonas |
本文献已被 SpringerLink 等数据库收录! |
|