摘要
目的潮霉素磷酸转移酶基因(hpt)是转基因作物中广泛应用的抗生素标记基因,本研究构建该基因的融合表达载体,以期得到足量纯化的HPT蛋白,为该外源蛋白的进一步安全性评价奠定基础。方法将hpt基因克隆到线性表达载体pET41 EK/LIC中,得到该基因的融合表达载体pET41EK/HPT,将pET41EK/HPT载体转入大肠杆菌BL21(DE3)中进行表达,沉淀中的包涵体进行了稀释复性、柱上复性和N-十二烷基肌氨酸钠(SKL)溶解稀释复性等复性研究及活性测定、标签切除、N端测序等工作。结果融合蛋白主要以无活性的包涵体形式存在,各复性方法表明SKL溶解稀释复性得到的蛋白在活性及产率方面明显高于其他两种方法,利用该方法每升培养物可获得78mg活性HPT融合蛋白,纯度约为93%,经肠激酶切割后,得到的蛋白与天然HPT蛋白N端氨基酸序列完全相同。结论利用本研究方法可得到在活性、分子量及氨基酸序列等方面与天然HPT蛋白一致的目的蛋白。
Objective Hygromycin B Phosphotransferase gene （hpt） is a widely used antibiotic selectable marker gene in the production of genetically engineered crops, Fusion expression vector of the hpt gene was constructed in order to obtain enough HPT protein for further safety assessment of the protein. Methods The hpt gene was cloned into liner expression vector pET41 EK/LIC and fusion expression vector pET 41EK/HPT was obtained, The constructed vector was transformed into expression host E. coil BL21 （DE3）, After induction, the expressed fusion protein was mainly aggregated as inclusion bodies. To get the active protein, dilution refolding, chromatographic refolding and Sarkosyl resolution and dilution refolding methods were explored, Then the fusion protein was digested by enterokinase and N-terminal amino acid sequence was conducted. Results The refolding study showed the protein got from Sarkosyl resolution and dilution refolding method had higher yield and bioactivity than those from other two methods. From this method 78mg active fusion obtained protein could be obtain from 1L bacterial culture and the purity was about 93% . From N terminal amino acid sequence it was confirmed that after digested by enterokinase, the HPT protein had same N-terminal amino acid sequence as the native HPT, Conclusion The results of this study illustrated that large quantity HPT protein could be acquired through prokaryotic host expression which had comparable molecular weight, N-terminal amino acid sequences and biological activities with those of native HPT protein.
出处
《卫生研究》
CAS
CSCD
北大核心
2007年第1期26-30,共5页
Journal of Hygiene Research
基金
国家科技部"863"计划(No.2004AA21221)
国家科技部973计划(No.2001CB109007)
关键词
潮霉素磷酸转移酶
安全性评价
融合蛋白
包涵体
复性
hygromycin B phosphotransferase gene, safety assessment, fusion protein, inclusion body, refolding
