Pyridoxal 5′-phosphate inhibits DNA binding of HNF1

Abstract

An efficient Escherichia coli expression system was constructed for the production of a variant form of HNF1 protein having the additional five amino acid residues (Asp-Arg-Trp-Gly-Ser) at the NH 2-terminal. The cDNA encoding HNF1 was ligated to 6×His tag and inserted into an inducible bacterial expression vector pRSET A. After expression in E. coli, the recombinant product was purified by Ni-NTA affinity column chromatography. The purified product showed expected NH 2-terminal sequence and specific binding to the HNF1 site. The effect of pyridoxal 5′-phosphate and its analogues on the binding activity of recombinant HNF1 was examined and found that only pyridoxal 5′-phosphate effectively inhibited the DNA binding. The concentration of pyridoxal 5′-phosphate that inhibited 50% of DNA binding was around 100 μM. Furthermore, we identified Lys197 of HNF1 molecule as the essential residue of DNA binding. These observations suggest that pyridoxal 5′-phosphate directly interacts with tissue-specific transcription factor HNF1 and modulates the binding to DNA.