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胡萝卜抗冻蛋白在大肠杆菌中的高效表达
引用本文:张党权,刘兵,范云,何炎明,王金发.胡萝卜抗冻蛋白在大肠杆菌中的高效表达[J].中山大学学报(自然科学版),2003,42(3):51-55.
作者姓名:张党权  刘兵  范云  何炎明  王金发
作者单位:中山大学生命科学学院∥教育部基因工程重点实验室,广东,广州,510275
基金项目:广东省自然科学基金资助项目 ( 990 2 5 8),科技部植物转基因重大专项资助项目 (J0 0-A-0 0 9)
摘    要:以pET—11a为载体构建了胡萝卜抗冻蛋白非融合表达的重组质粒,并在大肠杆菌菌株BL21(DE3,pLysS)中成功地进行了高效表达。表达的蛋白质以包涵体的形式存在,相对分子质量为36800。经正交试验得到了优化的诱导表达条件。包涵体经各种缓冲液洗涤纯化、溶解、复性和超滤浓缩后,经SDS—PAGE电泳检测,纯度达单一带水平。生物学活性研究表明,重组表达的胡萝卜抗冻蛋白具有很强的热滞活性和提高细菌耐寒性的作用。

关 键 词:胡萝卜Daucus  carota  var.sativa  抗冻蛋白  表达  包涵体  诱导  复性
文章编号:0529-6579(2003)03-0051-05
修稿时间:2002年7月15日

High Efficient Expression of Carrot Antifreeze Protein inE.coli
ZHANG Dang-quan,LIU Bin,FAN Yun,HE Yan-ming,WANG Jin-fa.High Efficient Expression of Carrot Antifreeze Protein inE.coli[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2003,42(3):51-55.
Authors:ZHANG Dang-quan  LIU Bin  FAN Yun  HE Yan-ming  WANG Jin-fa
Abstract:Using pET-11a as vector, a recombinant plasmid containing coding seque nce of carrot antifreeze protein(AFP) was constructed and high efficiently expre ssed in the E. coli BL21(DE3, pLysS) strain. A non-fusion expression protoc ol was adopted to express carrot AFP, the expressed carrot AFP existed in the fo rm of inclusion body in the total proteins, and the molecular weight of the reco mbinant protein is 36 800. An optimal inductive condition was obtained by a desi gned orthogonal experiments protocol. The AFP inclusion body was dissolved after washed with kinds of washing buffers, and the AFP in the supernatant was refold ed and concentrated by ultrafiltration. The purified AFP displayed a single band by SDS-PAGE analysis, and could increase low temperature resistance of bacteria with thermal hysteresis activity.
Keywords:carrot(Daucus carota var  sativa)  antifreeze protein  expression  i  nclusion body  induction  renaturation
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