Overexpression, Purification And Site-Directed Mutagenesis Of Two Thioredoxions From Maize

Thioredoxins are universally distributed in living things with the activity of disulfide reductases. They are main transmitters in the redox signal in the cells and play the important roles in the modification of the protein post-translation. The richest types of Trxs have been discovered in cytosol, mitochondrion and chloroplast of higher plants. Several types of Trxs, named Trx-f, Trx-m, Trx-x and Trx-y, have been studied in chloroplast. Among them, Trx-f and Trx-m mutant were used for screening the associated proteins in chloroplast. About 90 proteins have been determined in C3 plants. The properties of Trx-f and Trx-m in chloroplast have been reported with the molecular mass of about 12 kD from A. thaliana and spinach. They contain the conserved active site of WCGPC sequence. They play the function by disulfide/dithiol interchange reaction through the two cystine residues. There is no literature on Trx from C4 plants. In this study, we cloned two genes encoding Trx-f, Trx-m from maize, and purified recombinant Trx expressed in Escherichia coli. The site-directed mutations for two Trxs were conducted. The results are as follows:1. The amino acid sequences from maize published in Genbank were compared by Clustal W. The most homologous Trx-f and Trx-m were selected. The signal peptides from both types of Trxs were analyzed by ChloroP program. The first amino acid residues for Trx-f and Trx-m are Ser54 and Ser58 respectively. The restriction enzyme sites in the genes encoding Trx-f and Trx-m were analyzed using NEBcutter. Total RNA were extracted from young leaves of maize inbred lines B73. The one-step RT-PCR amplifications were carried out. The amplified product of about 400 bp was detected by agrose electrophoresis and inserted into T vectors. Sequencing analysis confirmed the sequence identical to the published Trx genes, compared by NCBI Blast.2. Two cloned Trx genes cut with BamH I and Hind III respectively were inserted into the individual expression vector pQE80 with the same treatment. The recombinant vectors were transformed in E.coli DH5αand induced for about 5h with IPTG at the final concentration of 0.5mmol/L. The recombinant proteins were purified using Ni-NTA chromatography with the extra amino acid residues MRGSH6G. Site-directed mutations were carried out using the mutation primers based on the coding analysis using Translate tool. The second cystine residue in active site of Trx was replaced by serine in Trx-f or by alanine in Trx-m. The mutated sites were identified by the gene sequencing. The mutated Trxs were also overexpressed and purified. The wild type and mutated protein were analyzed by SDS-PAGE with the molecular weight about18kD,14 kD. 3. The above-mentioned two gene fragments cut with BamH I and Hind III respectively were also inserted into the expression vector pSUMO with the same enzyme excise. The recombinant vectors were transformed in E.coli BL21(DE3) respectively. The Trx fused the histidine-tagged SUMO at N terminus were overexpressed and purified to apparent homogeneity. The proteinase Ulp, specific cleaving SUMO protein, was also purified. Using Ulp proteinase, the SUMO fusion was removed and Trx-f and Trx-m was released with an extra glycine residue. The purified Trxs will facilitated the functional analysis.In general, we purified the wild type and mutated Trx-f and Trx-m, which will help to analyze their functions and expression in maize leaves, to detect interactions and molecular regulating mechanism between the target protein, and to screen the target protein in maize chloroplast.