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 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 11,817 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.
Stein
M
M
Laboratoire de Physiologie Végétale Moléculaire, URA 1128 CNRS, Université de Paris-Sud, Orsay, France.
Jacquot
J P
JP
Jeannette
E
E
Decottignies
P
P
Hodges
M
M
Lancelin
J M
JM
Mittard
V
V
Schmitter
J M
JM
Miginiac-Maslow
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D054479:Chloroplast Thioredoxins; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D013879:Thioredoxins