Escherichia coli cells JM109 harboring the pMU388 plasmid encoding for Cry4Ba and its mutants R158A (12) , R158E, R158Q (13) , Y170A and Y170F (14) (link) were grown at 37℃ in Luria-Bertani medium containing 100 μg/ml ampicillin. Protein expression and production of toxin inclusions were performed as previously described (9) (link). Protoxin inclusions (130 kDa) were solubilized in 50 mM sodium carbonate (pH 10.0) and digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma-Aldrich, St. Louis, MO), resulting in a 65 kDa of the activated toxin, which was subjected to protein purification by gel filtration chromatography equipped with a Superose 12 column (GE Healthcare, Uppsala, Sweden). The purified 65-kDa protein was resolved by (12% gel) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands of the 47 kDa and the 18-20 kDa were then recovered by electroelution.
L 1 tosylamido 2 phenylethyl chloromethyl ketone treated trypsin
Manufactured by Merck Group
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin is a laboratory enzyme used in protein research and analysis. It is a modified version of the proteolytic enzyme trypsin, which is used to cleave proteins at specific amino acid residues. The chloromethyl ketone treatment of trypsin alters its specificity and activity.
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3 protocols using l 1 tosylamido 2 phenylethyl chloromethyl ketone treated trypsin
1
Production and Purification of Cry4Ba Toxin
2
Influenza Virus Infection Kinetics
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Monolayers of A549 cells were inoculated at a multiplicity of infection of 0.01 50% tissue culture infectious dose (TCID50) with DK/BD821/09 (H10N7), DK/BD8988/10 (H10N9), DK/BD24035/14 (H10N1), and Dk/BD24268/15 (H10N6). Cells were incubated at 37 °C in RPMI containing 0.2 µg/mL of L-1-tosylamido-2-phenylethyl chloromethyl ketone–treated trypsin (Sigma-Aldrich, St. Louis, MO). Supernatants were collected at different time points and titrated in MDCK cells by TCID50.
3
Proteolytic Clot Digestion and Mass Spectrometry
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Our protocol was adapted from Schmitt et al. ]. Clots were treated with hydroxylamine solution (1 M NH 2 OH-HCl, 4.5 M Gnd-HCl, 0.2 M K 2 CO 3 ; pH 9) at 45 °C on a vortex shaker for 16 hours. Following incubation, the samples were centrifuged at 17 000 g for 1 minute and the supernatant was removed. Clots were stored at -80 °C until ready for analysis. Prior to mass spectrometry analysis, clots were subjected to proteolytic digestion. Clots were washed with 100 mM triethylammonium bicarbonate buffer (TEAB; pH 8.5) prior to centrifugation at 17 000 g for 2 minutes at 4 °C and removal of the supernatant. Proteolytic digestion was performed using 25-μg PTMScan Lys-C Protease (Cell Signaling Technology) and 5 μg L-1-tosylamido-2-phenylethyl chloromethyl ketone treated trypsin (Sigma Aldrich) in 100 mM TEAB at 300 rpm for 16 hours at 37 °C. A further 5 μg L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin was added, and the digestion continued for another 2.5 hours.
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