Expression And Functional Analysis Of The Proteins Of Ferredoxin/thioredoxin System From Maize

There are two redox signals systems in photosynthesis of plant Chloroplast, One of the major systems are ferredoxin/thioredoxin system, which is composed of three proteins, thioredoxin (Trx), ferredoxin: thioredoxin reductase (FTR) and ferredoxin (Fd). So far, only arabidopsis thaliana ferredoxin/thioredoxin system has been functionally analyzed. In this study, we analyzed the function of three recombinant proteins from Escherchia coli.The main results are as follows:1. The purified thioredoxins displayed pI values of 4.6 for Trx-m and 5.9 for Trx-f. The modification of the proteins by the specific cystine reagent AMS revealed that the purified wild type thioredoxins displayed the redox states, but the mutated thioredoxins showed the reduced state, suggesting that there is no disulfide bond in the mutated thioredoxins. The reduction of insulin suggested that the m type thioredoxin has more active than f type thioredoxin. Both protein mutants hardly reduced insulin. The entrapped proteins from maize young leaves by the immobilized f type thioredoxin mutant were more diverse than those by the mutated m type thioredoxin.2. The genes encoding the mature FTR variable subunit and catalytic subunit were also amplified. The FTR subunits were overexpressed in Eecherchia coli and purified. All of them displayed one band on. The molecular weight was estimated 11 kD for FTRA, FTRB for 14 kD, as denoted by SDS-PAGE. The purified recombiniant FTR variable subunit was stable by the deletion of the first 20 amino acid residues.3. The amplification of gene encoding the maize Fd1 was carried out. The purified Fd1 is about 12 kD and displayed brown colour with the absorption peak of 315 nm, 415 nm and 459 nm by UV-visible spectra scanning. The [2Fe-2S] cluster was identified by EPR experiments. The proteins interacting with Fd were also detected.In general, the recombinant proteins involved in ferredoxin/thioredoxin system from maize were purified and characterized. These proteins will be applied in the analysis of the target proteins regulated by thioredoxin in several cereals. The proteins and their mutants fused with His-tag at N terminus were anchored on Ni-NTA and used to entrap the bound target proteins from maize leaves.