Design and construction of pET32b(+)Rh expression vector based on pET system to facilitate purification.

Purification of proteins is an essential step to determining their function and structure. Proteins are often combined with a tag to facilitate purification prosses. The aim of this study was to design and construct pET32b(+)Rh vector based on pET-32b(+) expression vector to simplifiy purification of recombinant proteins only by Nicle column. The primer design was performed taking into account the targeted changes. Using PCR reaction, the desired fragment was amplified and cloned in pET-32b(+) vector. The recombinant vector was transformed into Escherichia coli. After screening by colony-PCR and restriction enzyme analysis, sequencing was performed. Vector function study, was performed by investigating the expression and purification of a toxic peptide IOD-NaTx with disulfide bonds in pET32b(+)Rh vector. In new pET32b(+)Rh vector, the histidine tag site at the C- terminal of thioredoxin was removed and the N-terminal of the recombinant peptide would not change much if BamHI is used for cloning . To evaluate the function of the vector, the soluble expression of a toxic peptide was successfully determined and two-step purification using IMAC method resulted into the pure recombinant peptide. In this study, the simple and fast method of cloning was used to construct a modified expression vector using a commercial plasmid. The new pET32b(+)Rh vector can be used to express complex peptides with disulfide bonds fused with thioredoxin. The recombinant protein could be purified simply without need to costly methods such as HPLC and FPLC or other chromatography columns.