[25] Analysis and manipulation of target enzymes for thioredoxin control

Publisher Summary This chapter deals with the purification of the protein from the leaves of C 4 plants (in which the enzyme is present at a much higher level relative to C 3 plants), the optimal conditions for its reductive activation, and activity measurements. Other procedures have been published for the purification of the protein from C 4 or C 3 plants. A system is described for the expression and purification of the enzyme from Escherichia coli cells and for its modification to make it even more versatile. The purification procedure using plant leaves is time-consuming and requires large amounts of plant material. Thus, it is often more convenient to overproduce the protein in E. coli . The chloroplastic thioredoxin-dependent enzymes are nuclear-encoded. Hence, their full-length cDNA sequence comprises a transit peptide which is processed after protein import into the chloroplast. Protein purification is carried out essentially as for the plant enzyme. However, the overproduction of the enzyme by transformed bacteria allows some simplification of the procedure. The mutated protein, overproduced in E. coli , is purified essentially by following the same procedure as for the wild-type protein. However, it binds more strongly to the Matrex red A affinity column.