克雷伯杆菌甘油脱氢酶基因的克隆表达与纯化.pdf

生 物 工 程 学 报 Chin J Biotech 2008, March 25; 24(3): 495-499 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究简报 张婷婷, 方柏山, 王耿, 王飞飞 , 362021 : 以克雷伯杆菌(Klebsiella pneumoniae)基因组 DNA 为模板, 运用 PCR 扩增得到编码甘油脱氢酶(GDH)的基因 (gldA), 并克隆到 pMD-18T 载体上, 构建克隆载体 pMD-gldA 。经测序正确后, 将 gldA 亚克隆至表达载体 pET-32a(+) 上构建表达质粒pET-32gldA 。在乳糖诱导下, 携带pET-32gldA 的E . coli BL21 (DE3) 高效表达分子量约为54 kD 的可溶 性蛋白。表达产物带有His6-tag 标记, 选用Ni 柱对表达产物进行纯化, 纯化后酶液的比活为 188 u/mg, 纯化倍数和回 收率分别为3 倍和67.5% 。 : 甘油脱氢酶(GDH), 克雷伯杆菌, 蛋白纯化 Cloning Expression and Purification of Glycerol Dehydrogenase from Klebsiella pneumoniae Tingting Zhang, Baishan Fang, Geng Wang, and Feifei Wang Key Laboratory of Industrial Biotechnology Fujian Province, Huaqiao University, Quanzhou 362021, China Abstract: The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDSgel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%. Keywords: glycerol dehydrogenase, Klebsiella pneumoniae, protein purifica