Interaction of Recombinant Human Regulatory Factor X, 6 DNA Binding Region with Insulin Promoter in vitro

• Main Authors: Shruti Balaji, Anasuya Ganguly
• Format: Article
• Language: English
• Published: Applied Science Innovations Private Limited 2016-12-01
• Series: Carbon: Science and Technology
• Subjects: transcription factor; recombinant protein expression; electrophoretic mobility shift assay; insulin; diabetes; RFX6
• Online Access: http://www.applied-science-innovations.com/cst-web-site/CST-8-4-2016/CST-228-8-4-2016-48-62.pdf

The transcription factor, Regulatory Factor X, 6 (RFX6), is almost exclusively found in pancreatic islets where it maintains the functional identity of β-cells. It regulates insulin secretion directly by binding to the insulin promoter and indirectly by altering the expression of components of the voltage-gated ion channels. In order to better understand the mechanics of this binding, the present study describes the over expression and purification of the human RFX6 DNA binding region (RFX6 DBR) in a prokaryotic system. Using the pET28a(+) vector and E.coli host systems, a C-terminal His-tagged, 11kDa recombinant RFX6 DBR was expressed and purified. The identity of the protein was confirmed by Western blot analysis. The RFX6 DBR is able to bind to a 450 bp insulin (INS) promoter segment in vitro with an approximately 4x108 molar excess of protein required for the short-lived binding of protein to DNA. The basic molecular characterization of the RFX6 DNA binding region whose mutation causes certain forms of neonatal and type 2 diabetes, is reported here for the first time. The present study could have implications in understanding RFX6 as a target of pharmacological intervention.