title: Enhanced expression and purification of inositol 1,4,5-trisphosphate 3-kinase A through use of the pCold1-GST vector and a C-terminal hexahistidine tag in Escherichia coli.
abstract: Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A, alternative name: ITPKA) is a neuron-specific enzyme that converts 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) through its kinase domain. In addition, transient overexpression of IP3K-A induces morphological changes in dendritic spines of excitatory synapses in a kinase-independent manner, apparently by modulating the organization of the neuronal cytoskeleton. Although the procurement of a purified recombinant IP3K-A protein would be indispensable for the biochemical elucidation of its physiological roles, production of recombinant IP3K-A has proven technically challenging in conventional Escherichia coli expression systems. These difficulties stem from low enzyme solubility, as well as poor protein quality caused by the tendency of IP3K-A to split into partial fragments. In present study, we newly introduced cold-shock expression vector (pCold1) together with a C-terminal hexahistidine tag (C-HIS) to enhance the expression levels of recombinant IP3K-A in E. coli. Importantly, when compared with other commonly-employed bacterial expression systems, the pCold1 system improved the yield and the purity of full-length IP3K-A due to the exclusion of truncated enzyme forms, and also enhanced the solubility of the enzyme. Furthermore, the functional integrity of purified IP3K-A was confirmed in both kinase activity assay and microtubule binding assay. Recombinant IP3K-A acquired via this modified protocol will be expected to facilitate the exploration of the enzyme's biochemical profile, both structurally and functionally.
authors.affiliation: Department of Anatomy, College of Medicine, Korea University, Brain Korea 21, Seoul 136-705, Republic of Korea.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Lee
authors.affiliation: Department of Anatomy, College of Medicine, Korea University, Brain Korea 21, Seoul 136-705, Republic of Korea.
authors.forename: Seungrie
authors.initials: S
authors.lastname: Han
authors.affiliation: Department of Anatomy, College of Medicine, Korea University, Brain Korea 21, Seoul 136-705, Republic of Korea.
authors.forename: Seungkyun
authors.initials: S
authors.lastname: Woo
authors.affiliation: Department of Anatomy, College of Medicine, Korea University, Brain Korea 21, Seoul 136-705, Republic of Korea.
authors.forename: Hyun Woo
authors.initials: HW
authors.lastname: Lee
authors.affiliation: Department of Anatomy, College of Medicine, Korea University, Brain Korea 21, Seoul 136-705, Republic of Korea.
authors.forename: Woong
authors.initials: W
authors.lastname: Sun
authors.affiliation: Department of Anatomy, College of Medicine, Korea University, Brain Korea 21, Seoul 136-705, Republic of Korea. Electronic address: kimhyun@korea.ac.kr.
authors.forename: Hyun
authors.initials: H
authors.lastname: Kim
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics*; D008870:Microtubules / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase
keywords: Bacterial expression system; Histidine-mediated affinity purification; Inositol 1,4,5-trisphosphate 3-kinase A; Recombinant protein; pCold vector