Expression, Purification, and Characterization of Human NBD1 Wild Type
University of Texas Southwestern Medical Center at Dallas
Philip J. Thomas Laboratory
Expression and Culture Conditions:
A 5mL Inoculum of BL21-DE3 cells containing the SMT-3 fusion in the pET 28 expression system was grown overnight in LB medium at 37oC with kanamycin (50ug/mL working concentration) present. The inoculum was split evenly (750uL) among 6, 2 liter Erlenmeyer flasks containing 1 liter of LB, and allowed to grow for 8 hours. The cells were induced with 0.179g of IPTG and cooled to 15oC overnight for 16 hours. The OD600 at the time of induction was 2.0. The cells were harvested in 1 liter centrifuge bottles, and pelleted at 4000 RPM, 4oC for 30 minutes. After decanting the supernatant each pellet was resuspended in 10mL of Lysis buffer (See Buffer Recipes). The resuspension was combined into 3, 50mL conical vials and lysed according to the purification protocol.
Purification of Human NBD1:
The cells were lysed by sonication (4x 1 minute 50% duty cycle and Output Control of 5). The lysate was centrifuged at 40,000g for 45 minutes to separate the soluble and insoluble fractions, and was loaded onto a pre-equilibrated 5mL bed of Ni Sepharose 6 Fast Flow resin (GE Amersham). The column was equilibrated with 5 column volumes (CV) Loading Buffer (See Buffer Recipes). During this step the elution tubes were prepared with ATP and 2-Mercapto Ethanol so that in 5mL fractions the concentration of each was 2mM. The sample was loaded and bound to the column, and then washed to baseline. The bound sample was then washed with 5 CV wash buffer (See Buffer Recipes), and the sample was eluted in 5 CV of Elution Buffer (See Buffer Recipes). For the greatest purity, the fractions that eluted in the wash step were discarded, and only the fractions in the peak of the elution, not the Imidazole trail, were collected. Samples were taken for SDS PAGE analysis, and then pooled together for concentrating. The Centrifuge used for concentration was the Beckman Coulter Allegra 6R with a swinging bucket rotor. Concentration was performed using the Amicon Ultra 15 30,000 MWCO centrifugal filters (Millipore). The protein was concentrated using 10-minute spins at 4000rpm, 4oC, with special care given to resuspending the glycerol gradient that occurred between each spin. The final volume after concentrating was less than 5mL. Next, the SMT-3 fusion was cleaved off of the NBD by using a 1:1000 dilution of ULP1 (Ubiquitin like protease) on ice for 1 hour. The protein was filtered using a Nalgene 0.22-micron syringe filter and injected onto a Hi Load 16/60 Superdex S200 prep grade gel filtration column (GE Amersham), and ran in S200 buffer (See Buffer Recipes). The void volume fractions were rejected and the protein was loaded back onto the Ni Affinity column to remove the his-tagged SMT-3. The flow through was collected and concentrated in the same manner as before, except in a 10,000 MWCO Amicon Ultra 15 (Millipore). The protein was filtered again and injected onto the Superdex gel filtration column for buffer exchange. The protein was then concentrated to 1mg/mL and aliquoted into 200uL fractions. Finally, the protein was frozen in liquid nitrogen and stored at –80oC until further analysis could be done.
Analysis and Characterization of Human NBD1:
Before any analysis was done the protein was thawed on ice and centrifuged for 30 minutes in an Amicon YM-100 centrifugal filter (Millipore) to remove any protein that aggregated and became insoluble. The flow through was collected and used for analysis. First, a 10% SDS polyacrylamide gel of the protein was ran with 1ug, 3ug, and 10ug loaded to check the purity of the gel (See Figure 1). Next, a fluorescence spectrum was collected from 300 to 400 nm with the excitation set to 280 nm. The sample was prepared by making a 5-fold dilution of protein in S200 buffer into Phosphate Buffer (See Buffer Recipes) to give a 5uM final concentration of protein (See Figure 2). Next, an aggregation assay was performed by monitoring turbidity at 222 nm between 2oC and 60oC at 5uM concentration (See Figure 3). Circular Dichroism spectra were also collected in the same manner, by a 5-fold dilution into phosphate buffer (See Figure 4). The reason for the dilution into phosphate buffer is to minimize the interference caused by salts, Tris and ATP. Finally, a post aggregation CD spectrum was collected to show the change in signal after the aggregation event (See Figure 5).
Figure 1 Coomassie stained gel to show purity of protein.
50mM Tris, 100mM L-Arginine, 50mM NaCl, 5mM MgCl2 hexahydrate, 12.5% Glycerol, 0.25 IGEPAL CA630, 2mM 2-Mercapto Ethanol, 2mM ATP, pH 7.6
20mM Tris, 500mM NaCl, 5mM Imidazole, 12.5% Glycerol, pH 7.6
20mM Tris, 500mM NaCl, 60mM Imidazole, 12.5% Glycerol, pH 7.6
20mM Tris, 250mM NaCl, 400mM Imidazole, 12.5% Glycerol, pH 7.6
50mM Tris, 150mM NaCl, 5mM MgCl2 hexahydrate, 2mM ATP, 2mM 2-Mercapto Ethanol, 12.5% Glycerol, pH 7.6
20mM NaPO4, 1mM DTT pH 7.4