High-throughput screening (HTS)
Malachite green-based ATPase assay was used to screen the natural
compounds library of our lab. The assay procedure was adopted from
previous reports with modifications indicated below (42 ).
Colorimetric determination of VCP ATPase activity was performed as
follows: 1 μM 6×His-VCP or corresponding fragments were incubated with
reaction buffer, which contains 100 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 20
mM KCl, 0.01% Triton-X100. An aliquot (15 μl) of this mixture was added
into each well of a 96-well plate with 40μM compounds. After the
incubation for 1hr at 4°C, then 10 μl ATP was added into each well to
start the reactions. After the incubation of the reaction mixture for 30
min at 37°C, an aliquot of 80 μl of malachite green reagent was added to
each well and 10 μl 34% sodium citrate was immediately used to stop the
hydrolysis of ATP. Samples were mixed thoroughly and incubated at 37 °C
for another 15 min. Absorbance of each well at OD620 was then measured
by a FlexStation3 (Molecular Devices). Calculations of enzymatic
activity is conducted by curve fitting (based on Michealis–Menten
model) using GraphPad Prism 5 software.
Plasmid construction, protein expression and
purification.
Human wild-type VCP protein (residues 1–806) and VCP fragment protein
including N domain (residues1-188), N-D1 domain (residues 1–481), D2
domain (residues 461–806) and D1-D2 domain (residues(188-806), were
subcloned into the pET-28a(+) vector, proteins were expressed in E. coli
BL21 (DE3) bacteria with His-tag .GST,GST-LC3 were expressed in E. coli
BL21 (DE3) bacteria with GST-tag.
For expression of recombinant his-tag proteins, E. coli BL21 (DE3)
containing the desired plasmid was grown in LB medium containing 50 μg/L
Kanamycin with shaking at 37 ℃ to an OD600 of 0.5~1.
Then cell culture was cooled down to 18 °C and induced with 1 mM IPTG
and harvested 16h later by centrifugation. Cells were lysed in a lysis
buffer (150 mM NaCl, 1% Troten-X-100, 50 mM Tris-HCl (pH 8.0), 20 mM
imidazole,5% glycerol,5 mM MgCl2,5mM,2mM β-mercaptoethanol , 0.1%
lysozyme and a protease inhibitor PMSF). The lysate was centrifuged at
10000 rpm for 30 min at 4 ℃, and the supernatant was loaded onto Ni-NTA
resin (QIAGEN) at 4℃ with rotation for 60 min and washed with a buffer
containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 20 mM imidazole,5%
glycerol,5 mM MgCl2. The recombinant proteins were eluted with a 50–250
mM imidazole gradient, concentrated by Centriprep (Millipore), and kept
in storage buffer (150 mM KCl, 50 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 20%
glycerol).
Recombinant GST-fused proteins were expressed in Escherichia coli,
purified with glutathione-Sepharose , and eluted with 25 mM glutathione,
and the buffers were exchanged into 150 mM KCl, 50 mM Tris-HCl (pH 7.5),
1 mM MgCl2, 20% glycerol with Desalination column. Protein
concentrations were determined by Pierce®BCA Protein Assay Kit (Thermo)
and protein purity was monitored by SDS-PAGE and Coomassie blue
staining.
Partially proteolysis
assay
Trypsin digestion was performed in storage buffer (50 mM Tris-HCl, pH
7.0, 150 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA). VCP (10μM) and
compounds (15μM, final DMSO concentration 1%) were incubated for 1 h at
37 ℃ in a total volume of 50μl. DMSO (1 μl) was instead added as a
control. The reaction was started by the addition of trypsin , and
samples were incubated at 37℃. Reaction was immediately stopped by the
addition of 10μl of 6XSample buffer and heating at 100℃ for 10 min.
Digestion products were separated on 10% SDS-PAGE gels, and the
obtained bands were visualized by Coomassie blue staining.