Isolation and characterization of thioredoxin h from poplar xylem

Redox-dependent regulation based on disulphide/dithiol exchange reactions has been extensively studied in herbaceous plants, but up to now, there is no information concerning these systems in trees. Based on existing ESTs, a cDNA coding for a thioredoxin h has been isolated from a xylem poplar cDNA library. The nucleotidic sequence of poplar thioredoxin h displays significant homology to other thioredoxins h isolated from plants. It shows a variation in the active site with the sequence WCPPC instead of the more canonical WCGPC sequence found in most thioredoxins. The cDNA sequence has been introduced in an expression plasmid (pET3d) in order to express the corresponding recombinant polypeptide. The protein has been expressed to a high level and purified from Escherichia coli cells with a very high yield. Several of the physical and kinetic characteristics of this redox protein are described and found to be similar to other thioredoxin h. On the other hand, its stability to heat denaturation, is very different from those of other thioredoxins h characterized so far.