title: Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.
abstract: 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.
authors.affiliation: Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Liu
authors.affiliation: State Key Laboratory for Gene and Cell Therapy, Sichuan University, Chengdu, China.
authors.forename: Anliang
authors.initials: A
authors.lastname: Huang
authors.affiliation: Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Luo
authors.affiliation: Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
authors.forename: Haipeng
authors.initials: H
authors.lastname: Liu
authors.affiliation: Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
authors.forename: Huzi
authors.initials: H
authors.lastname: Han
authors.affiliation: Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: 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; Clover Biopharmaceuticals, Chengdu, China; GenHunter Corporation, 624 Grassmere Park, Nashville, TN 37211, USA. Electronic address: liangpeng@scu.edu.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Liang
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D011088:DNA Ligases
keywords: AK-T4; Adenylate kinase; Blue Sepharose; His-tag; T4 DNA ligase; adk