Expression of biologically active TAT-fused recombinant islet transcription factors.

AIMS

Differentiation of pancreatic endocrine cells depends upon the activation of genes that are regulated by islet transcription factors (ITFs). Evidence suggests that ITFs contribute to the development of the pancreas. These studies are focused on developing a system to deliver individual ITF from different stages of islet cell development to promote precursors or other cell types to trans-differentiate into islet-like insulin-positive cells.

MAIN METHODS

Protein transduction domains (PTDs) derived from the HIV-TAT peptide (YGRKKRRQRRR) were fused with three ITFs, Ngn3, Pdx1, and NeuroD/β2, to facilitate the uptake of ITF recombinant proteins into various cell types. The three TAT-fused ITFs, Ngn3, Pdx1, and NeuroD/β2 were constructed in a bacterial 6×His affinity tag-TAT recombinant protein expression system. The recombinant proteins were expressed using IPTG induction and purified to homogeneity using a nickel affinity column.

KEY FINDINGS

The biological activity of each TAT-fused ITF was demonstrated by nuclear translocation, induction of target gene promoter activity, and the trans-differentiation of pancreatic acinar cells, AR42J, into insulin-positive cells.

SIGNIFICANCE

This study provides advanced information for developing strategies using recombinant TAT-fused ITF proteins in place of adenoviral vectors for the conversion of pancreatic exocrine cells into insulin-positive cells for the treatment of diabetes.