Liu Xinxin, Huang Anliang, Luo Dan, Liu Haipeng, Han Huzi, Xu Yang, Liang Peng
Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
State Key Laboratory for Gene and Cell Therapy, Sichuan University, Chengdu, China.
Protein Expr Purif. 2015 May;109:79-84. doi: 10.1016/j.pep.2015.02.010. Epub 2015 Feb 17.
The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.
20世纪60年代T4 DNA连接酶的发现对分子生物技术的传播起到了关键作用。这种酶在全球生物医学研究中常规进行的重组DNA操作中已变得无处不在。为满足全球需求，人们付出了巨大努力来表达和纯化T4 DNA连接酶，但在大肠杆菌中可溶性T4 DNA连接酶的过量表达一直很困难。在此，我们探索利用腺苷酸激酶来增强T4 DNA连接酶的表达及其下游纯化。可在大肠杆菌中高水平表达为活性形式的大肠杆菌腺苷酸激酶，被融合到T4 DNA连接酶的N端。所得的带有His标签的AK-T4 DNA连接酶融合蛋白在大肠杆菌中大量过量表达，并通过由Blue Sepharose和Ni-NTA层析组成的两步纯化步骤，很容易纯化至近乎同质。纯化后的AK-T4 DNA连接酶不仅对DNA连接具有完全活性，而且由于腺苷酸激酶将ADP转化为ATP和AMP，除了ATP外还可使用ADP作为能量来源。因此，腺苷酸激酶可用作溶解性标签，以促进重组蛋白的表达及其下游纯化。
