Tarocystatin is mainly expressed as one of the abundant proteins in the periderm of mature corm and the mature corm inside.Expression of protease inhibitors in the periderm may defend against underground nematode and fungus attack or serve as storage proteins in the corm (Yang and Yeh ).For removing the GST-fusion protein, the column was loaded with 30 U of thrombin at room temperature overnight.
The complex of tarocystatin–papain was crystallized from a drop containing 15% polyethylene glycol monomethyl ether (PEG MME) 2000, 0.05 M sodium acetate trihydrate, p H 4.6, 0.1 M ammonium sulfate against a reservoir of 30% PEG MME 2000, 0.1 M sodium acetate trihydrate, p H 4.6, and 0.2 M ammonium sulfate (screening kit from Hampton Research Corp.).
The crystals of the complex appeared after approximately 1 month and disappeared after 6 weeks.
X-ray diffraction data were collected at 100 K and detected by a Quantum210 CCD detector at the BL13C1 beamline at NSRRC (Hsinchu, Taiwan).
Diffraction data were processed with HKL2000 (Otwinowski and Minor ) with the structure of papain (PDB ID:1PPN) used as the search model.
We resolved the crystal structure of the phytocystatin–papain complex at resolution 2.03 Å.
Surprisingly, the structure of the Nt D–papain complex in a stoichiometry of 1:1 could be built, with no Ct E observed.For the FL–papain complex structure, two obvious rotational and translational solutions for the papain structure were identified.The initial rigid body refinement of papain molecules gave an factor of 41.1%.Lyophilized papain was purchased as the commercial papaya latex enzyme (2× crystallized, Sigma-Aldrich, St Louis, MO, USA).The protein solution containing the FL tarocystatin and papain was prepared by mixing tarocystatin at 8 mg/ml with papain at 8 mg/ml in a 1:1 molar ratio.For the crystallization of the Ct E–papain complex, a mixture of protein solution was prepared of Ct E at 4 mg/ml with papain at 8 mg/ml in a 1:1 molar ratio.