作者:潘渠 胡福泉 陈恬 邱岳 王广科 李晋川
【摘要】 目的 在大肠杆菌中诱导表达以嗜酸乳杆菌克隆β半乳糖苷酶基因lacZ,为研究其酶学特性做准备。方法 从嗜酸乳杆菌ATCC 4356中克隆lacZ基因,并构建表达载体pET22blacZH、pQE31HlacZ和pQE31lacZ,然后在大肠杆菌中诱导表达,并测定表达产物的β半乳糖苷酶活性。结果 无标签的重组嗜酸乳杆菌β半乳糖苷酶LacZ获得了功能性表达,表达量可达2.28 kU/L.而融合HisTag的重组嗜酸乳杆菌LacZ却失去了β半乳糖苷酶活性,即使复性也不能恢复。结论 克隆表达的成功为该酶的酶学性质研究和可能的应用打下了基础。
【关键词】 嗜酸乳杆菌; β半乳糖苷酶; 克隆表达; HisTag; 复性
【Abstract】 Objective To clone and express the lacZ gene of L. acidophilus in E. coli for research of the properties of the putative βgalactosidase. Methods The lacZ gene was cloned from L. acidophilus ATCC 4356 and was inserted into three recombinant vectors: pET22blacZH, pQE31HlacZ and pQE31lacZ. The recombinant proteins were induced and were identified by SDSPAGE and βgalactosidase assay. Results The recombinant lacZ without tag was expressed as functional βgalactosidase, which yield was 2.28 kU/L, but the two recombinant LacZs with Histag were expressed as proteins without activity and its activity could not recovery by renaturation. Conclusion The study make a base for research of enzymatic properties and probable application in the future.
【Key words】 Lactobacillus acidophilus; βgalactosidase; expression; HisTag; renaturation
β半乳糖苷酶(βgalactosidase,EC 3.2.1.23)因为能水解牛奶中的乳糖而广泛的应用于乳品工业[1,2],又因为便利的显色反应而普遍应用于分子生物学研究[3,4]。寻找新的β半乳糖苷酶可以促进分子生物学研究和乳品工业发展。
最近嗜酸乳杆菌(Lactobacillus acidophilus)NCFM菌株基因组DNA(GenBank accession No. CP000033)已完成测序[5]。该菌基因组携带有3个推定的β半乳糖苷酶编码基因:lacA、lacZ和lacLM。已知lacLM基因编码的β半乳糖苷酶是该菌降解乳糖的主要酶[6,7],而lacZ基因却功能不明。迄今为止,还没有关于该基因编码产物的研究报道。嗜酸乳杆菌lacZ基因和著名的大肠杆菌(Escherichia coli)lacZ基因虽然同名,其序列却没有相似性。该基因相似于极端环境微生物的属于糖苷水解酶42家族(glycoside h [1] [2] [3] [4] [5] [6] 下一页 |