Mass Production and Characterization of Recombinant Human Grim-19 in Escherichia coli

Abstract

The gene associated with retinoid-interferon-induced mortality 19 (Grim-19, also known as NADH dehydrogenase 1 α subunit 13) induces apoptotic cell death in response to interferon-β (IFN-β) and retinoic acid (RA) treatment. The Grim-19/ Stat3 complex suppresses the essential genes that enhance cell growth. In addition, Grim-19 inhibits oncoproteins and viral gene products during tumorigenesis, which suggests that the development of biologics of Grim-19 could be considered. Although various cellular functions of Grim-19 were identified, limited information on the structural characteristics of Grim-19 is available. To study the structural characteristics of Grim-19 and to discover the possible biologics of Grim-19, the preparation of a large amount of Grim-19 is necessary. Grim-19 is the subunit of the mitochondrial membrane protein, ubiquinone, which is also found in the nucleus and cytoplasm. Thus, the stable recombinant protein of Grim-19 is not easily obtainable because of the aggregation of the protein. Here we successfully established an efficient expression system in E. coli by designing various E. coli vectors. Most of the E. coli vectors showed acceptable expression levels of Grim-19 in E. coli while they mainly produced an insoluble form of Grim-19 which could not be refolded sufficiently in vitro. Only the NusA-tagged Grim-19 fusion protein can be obtained as the soluble form in E. coli. The CD analysis showed Grim-19 mainly contains the α-helical structure with a melting temperature, 47℃.

Effect of Divalent Metal Ions on the Ribonuclease Activity of the Toxin Molecule HP0894 from Helicobacter pylori

“…The protein was purified using an open Ni 2+ -NTA column (Qiagen, Germantown, MD, USA), and the non-native hexahistidine tag was cleaved off with thrombin digestion at 20 • C for 12 h. Thrombin cleavage resulted in three non-native residues (GSH) attached to the N-terminus of HP0894. The protein was then subjected to gel filtration (Superdex 75 10/300 GL; GE Healthcare Sciences, Seoul, Republic of Korea) to achieve further purity [32].…”