Safarpour H, Safarnejad M R
Department of Microbial Biotechnology and Biosafety, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran.
Commun Agric Appl Biol Sci. 2012;77(3):145-50.
The plasmodiophoromycete Polymyxa betae, an obligate parasite of sugar-beet roots, is a natural vector of Beet necrotic yellow vein virus (BNYVV). 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 reverse transcriptase 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 expression and 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.
甜菜多黏菌是甜菜根的专性寄生菌，是甜菜坏死黄脉病毒（BNYVV）的天然传播介体。为开发针对包括甜菜多黏菌在内的任何病原体的基于蛋白质的诊断方法，必须制备一种特异性免疫原性蛋白。谷胱甘肽 - S - 转移酶（GST）在病原体生命周期的所有形态不同阶段均有表达，因此它是开发特异性抗体和用于诊断目的的免疫原性试剂的良好候选物。本研究描述了甜菜多黏菌GST蛋白的分离、克隆、大规模表达及纯化。为此，首先从受感染植物中分离总RNA，并以逆转录酶和寡聚 - dT引物以及mRNA为模板构建相应的cDNA。使用特异性引物从合成的cDNA中分离并PCR扩增编码GST的基因。扩增片段初步克隆到pTZ57R/T克隆载体中。经消化、PCR扩增及后续测序分析后，选择包含正确序列的完整克隆。接下来，回收具有正确序列的GST编码区并亚克隆到pET28a细菌表达载体中。重组蛋白在大肠杆菌BL21 - de3菌株中进行大规模表达，并通过含有Ni - NTA琼脂糖珠的柱在天然条件下通过固定化金属离子亲和色谱（IMAC）进行纯化。通过SDS - PAGE随后进行蛋白质印迹分析确认了成功的表达和纯化步骤。这些结果证实了约21 kDa的GST蛋白的高纯度和完整性。一般来说，培养基中纯化蛋白的总产量估计约为3.5 mg/mL。纯化后，鉴定出大部分纯化蛋白沉淀为过量的GST。为提高溶解度，将纯化蛋白的最终浓度降至0.5 mg/mL。
