ISOLATION, CLONING AND LARGE SCALE EXPRESSION OF GLUTATHIONE-S-TRANSFERASE (GST) PROTEIN OFPOLYMYXA BETAE

摘要

The plasmodiophoromycete Polymyxa betae, an obligate parasite of sugar-beet roots, is a natural vector of Beet necrotic yellow vein virus (BNYW). To develop protein based diagnosis for any pathogenic agents including P. betae, a specific immunogenic protein has to be prepared. The glutathione-S-transferase (GST) is expressed in all the morphologically different stages of the pathogen's life cycle, and then it is a good candidate as an immunogenic agent for developing of specific antibodies and diagnostic purposes. The present study describes isolation, cloning and large scale expression and purification of P. betae GST protein. For this aim, total RNA was initially isolated from infected plants and corresponding cDNA was constructed by using reversetranscriptase and oligo-dT primer as well as mRNA as a template. The gene encoding GST was isolated and PCR-amplified from the synthesized cDNA by using specific primers. The amplified fragments were preliminary cloned into pTZ57R/T cloning vector. Intact clone containing right sequence was selected after digestion, PCR amplification and subsequent sequencing analysis. Next, GST encoding region having right sequence was recovered and sub-cloned into pET28a bacterial expression vector. Large scale expression of recombinant protein was performed in BL21-de3 strain of E. coli and purification was carried out under native situation through Immobolized metal ion affinity chromatography (IMAC) in column containing Ni-NTA agarose beads. Successful expressionand purification steps were confirmed by SDS-PAGE followed by western blotting analysis. These results confirmed the high purity and integrity of GST protein which was around 21 kDa. Generally, the total yield of the purified protein in the culture medium was estimated at around 3.5 mg/mL. After purification, a major part of the purified proteins was precipitated identified as excess GST. To improve the solubility, the final concentration of purified protein was reduced to 0.5 mg/mL.