A rapid method for solubilization of chimeric human interferon regulatory factor-1 (IRF-1) protein in Escherichia coli.

Abstract

Interferon regulatory factor-1 (IRF-1) is a vertebrate transcription factor that plays significant roles in cell cycle regulation, anti-viral response, tumor suppression and immune response. High-level expression of recombinant IRF-1 at 37°C leads to the formation of insoluble aggregates (insoluble fraction) in Escherichia coli (E. coli), which usuallydevoid of biological activity. In this study, we use chemical additives such as mannitol, proline, L-arginine and CTAB (cetyl trimethly ammonium bromide)at the recommended concentration during cell lysis to aid in solubility at 37°C. The use of additives resulted in the increased solubility of the recombinant glutathione S-transferase-linked human IRF-1, with L-arginine being most effective. Here, we developed an efficient process for the manufacturing of soluble IRF-1 with the aid of minimizing the formation of degradation products and optimizing protein purification conditions. This result was further confirmed by western blot with anti-GST and anti-IRF-1 polyclonal antibodies. The functionality of GST-huIRF-1 was attained by elerophoretic mobility shift assay study as a clear band shifting showed with virus response element-Interferon beta (VRE-IFNβ) promoter region. Taken together, the biological activity of purified GST-huIRF-1 was also optimized and confirmed by supershift assay concluded that GST-huIRF-1 interacts with the VRE motif of IFNβ promoter that reflected to require for IFNβ gene regulation. We describe a straightforwardapproachfor theproductionofabsolutelysoluble and biologicallyactiveIRF-1 in E. coli. This method can be further used for the study of other recombinant proteins and this study will pave way for the analysis of IRF-1 function in vitro.