Purification and characterization of pea thioredoxin f expressed in Escherichia coli.
The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.
Hodges
M
M
Laboratoire de Physiologie Végétale Moléculaire, URA CNRS 1128, Université Paris-Sud, Orsay, France.
Miginiac-Maslow
M
M
Decottignies
P
P
Jacquot
J P
JP
Stein
M
M
Lepiniec
L
L
Crétin
C
C
Gadal
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D054479:Chloroplast Thioredoxins; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D015870:Gene Expression; D007544:Isopropyl Thiogalactoside; D008291:Malate Dehydrogenase / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000737:chemistry*; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D018724:Spinacia oleracea / Q000201:enzymology; D013879:Thioredoxins
D054479:Chloroplast Thioredoxins; D010940:Plant Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D013879:Thioredoxins; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D006597:Fructose-Bisphosphatase