A dicistronic construct for the expression of functional spinach chloroplast ferredoxin:thioredoxin reductase in Escherichia coli.

Ferredoxin:thioredoxin reductase (FTR) is a heterodimeric Fe&z.sbnd;S containing disulfide reductase involved in the light-dependent activation of photosynthetic enzymes. We have designed a dicistronic construct for the heterologous expression of this nucleus encoded chloroplast protein in Escherichia coli. The coding sequences for the two mature subunits have been inserted in tandem into the expression vector pET-3d. This dicistronic construct is correctly translated yielding soluble, perfectly functional FTR. The recombinant enzyme is composed of both subunits, contains the correctly inserted Fe&z.sbnd;S cluster as evidenced by its spectral properties and is indistinguishable from the enzyme isolated from leaves in its capacity to activate chloroplast fructose-1,6-bisphosphatase, one of the well known light activated enzymes of the Calvin cycle.