Recombinant human interferon regulatory factor-1 (IRF-1) protein expression and solubilisation study in Escherichia coli.

Abstract

Interferon regulatory factor-1 (IRF-1) is a tumor suppressor gene, which encodes a mammalian transcription factor that serves various vital functions in a cell, such as cell cycle regulation, immunomodulation, and antiviral response. We report full-length human IRF-1 cDNA cloning and expression in E. coli/BL21 cells with complete solubilisation of recombinant protein. We cloned the gene by the RT-PCR technique using ORF-specific primers followed by expression of recombinant IRF-1 in E. coli under GST fusion system. The profound expression of recombinant protein was observed after inducing with 0.5mM IPTG for 3h at 37°C. We observed few degradation products of low molecular mass along with full-length fusion protein. We successfully minimized the formation of low molecular mass degradation products of GST-huIRF-1 protein at 16°C. Simultaneously, we achieved the expression of recombinant protein in soluble fraction of E. coli/BL21 cells at 20°C with higher yield, which is crucial to the study of the biological functions of any protein. We further confirmed it by the immunoblotting technique using anti-IRF-1 and anti-GST antibodies under the induction of E. coli cells harboring the IRF-1 recombinant plasmid after sonicated and fractioned fractions. This work will serve as a platform for characterizing the recombinant protein that may pave the way to understand molecular mechanism of tumour suppression caused by this molecule.