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
- Authors
- Lee, Dongmin; Han, Seungrie; Woo, Seungkyun; Lee, Hyun Woo; Sun, Woong; Kim, Hyun
- Issue Date
- 5월-2014
- Publisher
- ACADEMIC PRESS INC ELSEVIER SCIENCE
- Keywords
- Bacterial expression system; Inositol 1,4,5-trisphosphate 3-kinase A; Histidine-mediated affinity purification pCold vector; Recombinant protein
- Citation
- PROTEIN EXPRESSION AND PURIFICATION, v.97, pp.72 - 80
- Indexed
- SCIE
SCOPUS
- Journal Title
- PROTEIN EXPRESSION AND PURIFICATION
- Volume
- 97
- Start Page
- 72
- End Page
- 80
- URI
- https://scholar.korea.ac.kr/handle/2021.sw.korea/98708
- DOI
- 10.1016/j.pep.2014.02.006
- ISSN
- 1046-5928
- 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 pColdl 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. (C) 2014 Elsevier Inc. All rights reserved.
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Collections - Graduate School > Department of Biomedical Sciences > 1. Journal Articles
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