Department Applied Biochemistry, Tokai University
○Yudai Ishii Tetsuo Takahashi Ryuta Mizutani
The protein splicing reaction has been exploited to allow peptide ligation or excision to proceed in vivo and in vitro. Miniaturization of the splicing domain would facilitate these protein splicing applications to the larger peptide targets. Crystallographic studies have been performed for recombinants of the VMA1-derived endonuclease intein (VDE) bearing N- and C-extein residues. The dodecapeptide-motif domain of VDE is responsible for the endonuclease activity and not required for the protein splicing reaction. This endonuclease domain is connected to the splicing domain with antiparallel beta strands. In the present study, the endonuclease domain is replaced with a beta-turn designed from the crystal structure, giving a minimum protein that undergoes splicing reaction.
Synthesized oligonucleotide fragments corresponding to the beta-turn were inserted to the HindIII - SacII site of the VDE allele of the pET expression vectors. The obtained VDEdelta463-699 with the wild-type sequence and VDEdelta463-699 bearing a mutation N737S were expressed and recovered from the supernatant of the Escherichia coli lysate. In the SDS-PAGE analysis, the VDEdelta463-699(wt) recombinant gave a product band, while VDEdelta463-699(N737S) gave a precursor band, indicating that the N737S recombinant is a minimum analog of the protein splicing precursor, and that the shortcut linkage forms an expected turn structure. The VDEdelta463-699(N737S) protein was purified by ammonium sulfate precipitation and cation chromatography procedures. Crystallization of the purified protein underway will lead to precise elucidation of the reaction mechanism of protein splicing.