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: Dongmin Lee, Seungrie Han, Seungkyun Woo, Hyun Woo Lee, Woong Sun, Hyun Kim
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 97 Pg. 72-80 (May 2014) ISSN: 1096-0279 [Electronic] United States
• PMID: 24576661 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• His-His-His-His-His-His
• Oligopeptides
• Recombinant Proteins
• Histidine
• Phosphotransferases (Alcohol Group Acceptor)
• Inositol 1,4,5-trisphosphate 3-kinase

Topics

• Cloning, Molecular
• Escherichia coli (genetics)
• Genetic Vectors (genetics)
• Histidine (genetics)
• Microtubules (metabolism)
• Oligopeptides (genetics)
• Phosphotransferases (Alcohol Group Acceptor) (chemistry, genetics, isolation & purification, metabolism)
• Recombinant Proteins (chemistry, genetics, isolation & purification, metabolism)
• Solubility