Creation and structure determination of an artificial protein with three complete sequence repeats

Shimizu, Rumi; Adachi, Motoyasu; Kuroki, Ryota; Blaber, M.

We have already succeeded in creation of the de novo designed protein (Symfoil) exhibiting the threefold symmetrical -trefoil fold based on the human acidic fibroblast growth factor. Based on the Symfoil protein, we created Symfoil-II having the three repeats. The Symfoil-II protein was expressed in Eschericha coli as soluble protein, and purified by metal affinity chromatography using nickel-chelated agarose. The Symfoil-II was further purified by anion-exchange column chromatography after removing the HisTag by proteolysis. Symfoil-II was crystallized in 0.1 M Tris-HCl buffer (pH7.0) containing 1.8 M ammonium sulfate as precipitant at 20C. The X-ray diffraction data was collected to 1.9 resolution using a Rigaku R-AXIS VII diffractometer. The crystal belongs to a monoclinic space group C2. The refined crystal structure of Symfoil-II showed three fold symmetry as is observed in other Symfoils.