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

Dongmin Lee, Seungrie Han, Seungkyun Woo, Hyun Woo Lee, Woong Sun, Hyun Kim

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 IP 3K-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.

Original languageEnglish
Pages (from-to)72-80
Number of pages9
JournalProtein Expression and Purification
Volume97
DOIs
Publication statusPublished - 2014 Jan 1

Keywords

  • Bacterial expression system
  • Histidine-mediated affinity purification
  • Inositol 145-trisphosphate 3-kinase A
  • pCold vector
  • Recombinant protein

ASJC Scopus subject areas

  • Biotechnology

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