摘要
目的探讨工程菌表达的耐热ＤＮＡ聚合酶的纯化方法。②方法收集ＩＰＴＧ诱导带有耐热ＤＮＡ聚合酶（ＴＤ聚合酶）基因表达质粒的工程菌株ＤＨ－ＴＤ４，用溶菌酶裂解，６０℃处理后，上清液用硫酸铵沉淀，根据溶解度差异进行粗分级，然后进行离子交换层析细分级，并经聚合酶链式反应（ＰＣＲ扩增）鉴定其活性。③结果纯化的ＴＤ聚合酶具有良好的聚合活性。④结论溶解度差异与离子交换层析是工程菌表达的耐热ＤＮＡ聚合酶纯化的主要步骤。
Objective To study the purification methods of heat resistant DNA polymerase expressed by engineering strain. Methods DHTD4 engineering strain with heat resistance DNA polymerase (TD polymerase) gene was induced by IPTG.Cells were collected by centrifugation and lysized with lysozyme. After the lysate was incubated at 60℃,protein in the supernatant was precipitated by ammonium sulphate, graded simply by solubility, and then futher purified by ion exchanger. PCR amplification was used to test the activity of the purified protein. Results The purified TD polymerase had fine activity. Conclusion Grading by solubility and purification of ion exchanger were the predorminant method in purification of protein expressed by engineering strain.The purified TD polymerase was usable in common PCR amplification.
出处
《青岛医学院学报》
1998年第4期235-236,共2页
Acta Academiae Medicinae Qingdao Universitatis
关键词
纯化
大肠杆菌
聚合酶链反应
耐热DNA聚合酶
deoxyribonuclease
ion exchange
purification
stability of enzyme
escherichia coli
