title: High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG.
abstract: Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine-5') pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake.
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University, Chengdu, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Luo
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University, Chengdu, China.
authors.forename: Caixia
authors.initials: C
authors.lastname: Wen
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University, Chengdu, China.
authors.forename: Rongchuan
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University, Chengdu, China.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University, Chengdu, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University, Chengdu, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Cui
authors.affiliation: Clover Biopharmaceuticals, Chengdu, China.
authors.forename: Joshua G
authors.initials: JG
authors.lastname: Liang
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University, Chengdu, China.|Clover Biopharmaceuticals, Chengdu, China.|GenHunter Corporation, Grassmere Park, Nashville, United States of America.
authors.forename: Peng
authors.initials: P
authors.lastname: Liang
mesh_terms: D000263:Adenylate Kinase / Q000235:genetics*; D000345:Affinity Labels / Q000737:chemistry; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D020224:Expressed Sequence Tags / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D006639:Histidine; D000263:Adenylate Kinase