Homology modeling, cloning, overexpression, and purification of GST-tagged full-length and C-terminal domain of Human Interferon Regulatory Factor 6
**Binita Kumari Sinha, Priyabrata Meher, Sanju Kumari Singh, Sindhuja Snehi, and *Tara Kashav
Department of Life Science, Central University of South Bihar, SH-7, Gaya Panchanpur Road, Village- Karhara, Post-Fatehpur, Gaya-824236 (India) Email: **binitasinha@cusb.ac.in, *tarakashav@cub.ac.in
ABSTRACT
Human Interferon Regulatory Factor 6 (Hu-IRF6) protein is a transcription factor, a member of the IRF family. IRF6 is involved in palatogenesis, gland development, and acts as a tumor suppressor. The purpose of the present study is to perform homology modeling to predict the structure, clone, overexpress and optimize the purification of IRF6 protein in native conditions. The full-length-IRF6 gene and CTD-IRF6 were cloned, overexpressed, and proteins were purified under nondenaturing conditions. Low temperature (16oC) with a low concentration of IPTG induction helps in the proper folding of protein which enhances protein solubilization. A significant improvement in protein solubility, stability, and yield was observed with additives like L-Arginine, ethylene glycol, LiCl, and fructose. The homology model shows the presence of a long linker connecting the NTD to CTD, this flexible linker region may be the reason for the less stability of full-length IRF6 protein. The addition of additives during purification is helpful to increase the IRF6 protein yield and stability in a soluble fraction which can be scale-up for large-scale purification of protein.