Expression and purification of thioredoxin-his6-ZmDREB2.7 fusion protein in Escherichia coli for raising antibodies

T. Nguyen, T. Nguyen, V. H. Nong, T. Huynh
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Abstract

Dehydration-responsive element-binding (DREB) proteins play a critical role in the plant’s drought-tolerance mechanism despite their presence in minor amounts in the cell. In this study, a maize-derived transcription factor protein, ZmDREB2.7, was overexpressed in the Escherichia coli strain Rosetta 1. The interested gene conjugating with the thioredoxin gene (TrxA) and his6 tag in the pET-32a vector encoded a 55.7 kDa fusion protein. The optimum condition for inducing the thioredoxin-his6-ZmDREB2.7 expression was five hours of induction with 0.05 mM IPTG at 300C. The Tris-HCl 20 mM pH 8.0 lysis buffer was harnessed to extract the recombinant protein for the purification process. Using the immobilized-metal affinity chromatography column, the recombinant protein was purified and then injected into rabbits. The antisera containing polyclonal antibodies (pAbs) could specifically recognize the ZmDREB2.7 fusion protein. This study represents updated data on the bacterial expression of the recombinant ZmDREB2.7 protein and the production of anti-ZmDREB2.7 pAbs.
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硫氧还毒素-his6- zmdreb2.7融合蛋白在大肠杆菌中的表达及纯化
脱水响应元件结合蛋白(DREB)在植物的抗旱机制中发挥着关键作用,尽管它们在细胞中含量很少。在本研究中,一种玉米来源的转录因子蛋白ZmDREB2.7在大肠杆菌Rosetta 1中过表达。在pET-32a载体中,与硫氧还蛋白基因(TrxA)和his6标签结合的基因编码了一个55.7 kDa的融合蛋白。诱导thioredoxin-his6-ZmDREB2.7表达的最佳条件为0.05 mM IPTG在300C下诱导5h。利用Tris-HCl 20 mM pH 8.0裂解缓冲液提取重组蛋白进行纯化。利用固定化金属亲和层析柱纯化重组蛋白,并将其注射到家兔体内。含有多克隆抗体(pAbs)的抗血清能够特异性识别ZmDREB2.7融合蛋白。该研究代表了重组ZmDREB2.7蛋白的细菌表达和抗ZmDREB2.7 pab的最新数据。
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