Engineering and Expressing an Insulin Promotor-Binding Fusion Protein
by Connor Fowler, Michael Wisniewski
Developed under the guidance of:
Dr. Taek You and Dr. Greg Buhrman
Biology
Maturity-onset diabetes of the youth type 4 (MODY4) is a form of type 2 diabetes (T2D) associated with missense mutations of pancreatic duodenal homeobox-1 (PDX-1), a para-Hox transcription factor necessary for pancreatic β-cell development, function, and survival. Musculoaponeurotic fibrosarcoma A (MafA), a tyrosine zipper homodimer, has also been linked to β-cell differentiation and function, and indicated in T2D pathogenesis. Both PDX-1 and MafA bind to sites on the proinsulin upstream promoter. A fusion protein of PDX-1, a flexible 26 amino acid linker region, and MafA was engineered, cloned, transformed into BL21DE3 competent cells, and expressed via IPTG induction. This construct will be used in DNA-binding studies with synthetic proinsulin upstream promoter DNA using electrophoretic mobility shift assays, culminating in solving the crystal structure of the promotor-construct complex using X-ray crystallography at NCSU. By understanding the DNA-protein interactions about the proinsulin upstream promoter, pathways to future T2D therapies targeting endogenous insulin production and β-cell function could be elucidated.
Contact
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Buies Creek, NC 27506 Directions
