Biochemistry & Analytical Biochemistry

Chimeric murine interferon regulatory factor-2 (IRF-2) binds on IRF-E (IRF binding Element), VREβ (Virus Response Element) but not binds on VREα

International Conference and Exhibition on Biochemical & Molecular Engineering

October 07-08, 2013 Hilton San Antonio Airport, TX, USA

Krishna Prakash and Pramod C. Rath

Accepted Abstracts: Biochem Anal Biochem

Abstract:

Background:IRF-2 is a multifunctional transcription factor having gene activation, repression and synergistic effect in conjunction with IRF-1. IRF-2 is also involved in type I IFN signaling by repressing INF β gene. So far, the molecular mechanism of its DNA binding activity remains elusive. Methods: We have done molecular sub-cloning, expression and EMSA study of chimeric murine IRF-2. Result: Here we report expression of chimeric murine IRF-2 as GST-IRF-2 fusion protein in E.coli/BL21 cells and demonstrated DNA binding activity by Gel retardation technique using radio 32 P labeled IRF-E motif (GAAAGT) 4 , Virus Response Element (VRE) of human INF β and IFN α gene. We observed five different masses DNA/GST-IRF-2 complexes (1-5) with IRF-E motif, three different masses DNA/GST-IRF-2 complexes (1-3) with VRE , but we couldnt observe any complex of DNA/GST-IRF-2 with VRE α . The specific binding on IRF-E motif was confirmed by carrying out 100-X fold cold competition with 32 P labeled IRF-E motif. In contrast to specific binding on VRE , we used negative control where we observed no binding complex, but we observed complexes with clones IPTG-induced extract. As far as binding on VRE α is concerned, we could not observe any complex in negative control as well as in IPTG-inducible clones extract. Conclusions: Chimeric IRF-2 binds to IRF-E motif and VRE β but not with VRE α . This study is first of its kind and paves the way to understand the differential DNA binding and molecular mechanism of DNA binding activity of the IRF-2 molecule, which is crucial for its function(s). Keywords: Interferon, Recombinant IRF-2, DNA binding domain, Gel retardation.