USTB-05-B基因在大肠杆菌中的表达与纯化研究

Research on the Expression and Purification of USTB-05-B in E.coli

采用生物学技术对高效降解线性微囊藻毒素-LR(microcystin-LR,MC-LR)的USTB-05-B酶进行克隆表达及纯化研究。首先利用分子克隆技术得到重组菌pET30a(+)/USTB-05-B/BL21(DE3)。在诱导剂IPTG浓度为0.25 mmol/L、温度30℃和诱导时间4 h条件下,重组菌能大量表达目的蛋白。然后利用亲和层析柱对重组菌破碎后的无细胞提取液(cell free extracts,CE)进行纯化,当咪唑浓度为100 mmol/L时,洗脱下来的蛋白纯度高。最终得到USTB-05-B蛋白纯度为91.1%,浓度为0.205 mg/m L。纯化后的USTB-05-B酶具有较高的活性,能在1 h内将10.8 mg/L的线性MC-LR降解完全。纯化后的目的蛋白为研究USTB-05降解MCs分子机理奠定基础,为有效提高降解MCs速率提供新材料。

Biological technology was used to cloning express and purify the USTB-05-B enzyme, who has the ability to high efficient degradation of linear microcystin-LR（MCs）. First of all, the pET30 a（＋）/USTB-05-B/BL21（DE3） recombinant bacteria was obtained with the molecular cloning technology. The recombinant bacteria could express lots of protein, when inducer IPTG concentration was 0.25 mmol/L, under the condition of temperature 30 ℃ and the induction time of 4 h. Then the cell free extracts were purified with affinity chromatography column. The high purity protein was eluted down, when the imidazole concentration was 100 mmol/L. The USTB-05-B protein concentration was 0.205 mg/m L, and the protein purity was 91.1%.Initial 10.8 mg/L of linear MC-LR could be completely eliminated within 1 hour by high purity USTB-05-B. The purified protein would lay a foundation to research on molecular mechanism of MCs degradation by USTB-05, and provide a new material to improve the rate of degradation of microcystins.