AY329081), which encodes the Cry8Ea1 protoxin, was constructed and stored by State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, the Chinese Academy of Agricultural Sciences (Shu et al., 2009b). The Superdex-200 columns were obtained from Amersham Pharmacia Biotech, and the Ultra centrifugal filters were from Millipore. DNase I (RNase-free) was purchased from Takara. Ultrapure guanidine hydrochloride (Gdm-HCl), proteinase K, TPCK-treated trypsin, α-chymotrypsin
from bovine pancreas, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and cholesterol were purchased from Sigma. All other reagents were local products of analytical grade. The B. thuringiensis HD8E strain http://www.selleckchem.com/products/Everolimus(RAD001).html was grown, and the protoxin was obtained as described previously (Guo et al., 2009a). Cry8Ea1 protoxin was treated
with DNase I at 4 °C for 12 h. Subsequently, the Cry8Ea1 protoxin was further digested separately INCB024360 molecular weight with trypsin (1 : 30 and 1 : 50 w/w) or chymotrypsin (1 : 30 and 1 : 50 w/w) at 37 °C for 1 h. Also, an aliquot of the Cry8Ea1 protoxin was treated with proteinase K (final concentration, 50 μg mL−1) at 37 °C for 1 h. The Cry8Ea1 protoxin and the products obtained after treatment with DNase I, DNase I/trypsin, DNase I/chymotrypsin, and proteinase K were fractionated by agarose gel electrophoresis on a 0.7% gel. The solubilized Cry8Ea1 protoxin was activated by digestion with chymotrypsin (1 : 50 w/w) at 37 °C for 1 h. The digested products were loaded on the Superdex-200 column (HR-10/30) through pre-equilibrated with 50 mmol L−1 Na2CO3 (pH 10.2) using a Pharmacia FPLC apparatus at a flow rate of 0.6 mL min−1.
A260 nm and A280 nm was monitored as the elution was being performed, and the peak fractions were collected. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and agarose gel electrophoresis. The Cry8Ea1 toxin–DNA complex was further treated with DNase I at 4 °C for 12 h. The products were then loaded onto the Superdex-200 column on the Pharmacia FPLC apparatus with the same buffer and parameters as above. The purified protein was analyzed by SDS-PAGE and agarose gel electrophoresis. The protein concentration was determined by the Coomassie blue protein dye-binding method (the Bradford method) with bovine serum albumin as the standard (Bradford, 1976). The unfolding experiments were performed at three different pH values in the following buffer systems: at pH 4.0 in 50 mmol L−1 acetic acid and 50 mmol L−1 H3PO4 adjusted with NaOH; at pH 7.0 in 50 mmol L−1 NaH2PO4 adjusted with NaOH; and at pH 11.0 in 50 mmol L−1 Na2HPO4 adjusted with NaOH. All buffers contained 150 mmol L−1 NaCl (Rausell et al., 2004).