INTRODUCTION
MATERIALS AND EQUIPMENT
1 Materials and equipment used in this protocol |
Materials or equipment | Source | Identifier |
a DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; b DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; c IPTG, Isopropyl β-D-1-thiogalatopyranoside; d PtdIns, L-α-phosphatidylinositol; e PMSF, Phenylmethylsulfonyl fluoride; f TAG, Triacylglycerol; g DTT, Dithiothreitol; h YT, Yeast extract-Tryptone; i HEPES, 4- (2-hydroxyerhyl) piperazine-1-erhanesulfonic acid | ||
Reagent | ||
dNTP Mix | GenStar | Cat#A113-01 |
DOPC a | Avanti Polar Lipids | Cat#850375 |
DOPE b | Avanti Polar Lipids | Cat#850725 |
DpnI | New England Biolabs | Cat#R0176 |
EasyPfu DNA Polymerase | TransGen Biotech | Cat#AP111-01 |
IPTG c | Amresco | Cat# N679-10G |
Liver PtdIns d | Avanti Polar Lipids | Cat#840042 |
OptiPhase Supermix Scintillation Cocktail | PerkinElmer | Cat#1200-439 |
Phusion High-Fidelity DNA Polymerase | New England Biolabs | Cat#M0530L |
PierceTM BCA Protein Assay Kit | Thermo Fisher Scientific | Cat#23225 |
PMSF e | Sigma-Aldrich | Cat#52332 |
T4 DNA Ligase | Takara | Cat#2011B |
TAG f | Extracted from rat fat pad in the laboratory | N/A |
TOP10 Competent Cell | TianGen | Cat# CB104 |
Transetta (DE3) Competent Cell | TransGen Biotech | Cat#CD801-02 |
Triolein [9, 10-3H(N)] | PerkinElmer | Cat#NET431001MC |
Tryptone | OXOID | Cat# LP0042B |
Yeast Extract Power | OXOID | Cat#LP0021T |
Buffers or solutions | ||
2× Sample Buffer | 100 mmol/L Tirs-HCl, 8% SDS (m/v), 20% glycerol (v/v), 0.2% Bromophenol blue (m/v), 200 mmol/L DTT g (pH 6.8) | N/A |
2× YT h Medium | 16 mg/mL Tryptone, 10 mg/ mL Yeast extract, 5 mg/mL NaCl | N/A |
Buffer B | 20 mmol/L HEPES i, 100 mmol/L KCl, 2 mmol/L MgCl2 (pH 7.4) | N/A |
Coomassie Brilliant Blue Dye Solution | 1 mg/mL Coomassie Brilliant Blue R-250, 45% CH3OH, 45% ddH2O, 10% CH3COOH | N/A |
Extraction Solution I | Methanol/chloroform/n-hexane=10:9:7 (v/v/v), stored light-protected at 4°C, prewarm solution before use | N/A |
Extraction Solution II | 0.1 mol/L K2CO3 (pH 10.5, adjusted with saturated H3BO3) | N/A |
Solution A | 0.25 mol/L Sucrose, 1 mmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L PMSF | N/A |
Tris-NaCl Buffer | 50 mmol/L Tris-HCl, 150 mmol/L NaCl (pH 7.4) | N/A |
Plasmid | ||
pET-28a-SMT3-N | Gift from Dr. Sarah Perret | N/A |
Equipment | ||
Centrifuge (5417R, 5424R) | Eppendorf | Cat#10148204 |
Gamma Meter | PerkinElmer | Cat#2470-0010 |
Vortex Oscillator | Scientific Industries | N/A |
Water Bath | Shanghai Yiheng Scientific Instrument | N/A |
Software | ||
Adobe Illustrator CS5 | Adobe | |
GraphPad Prism 7.0 | GraphPad Software | |
ImageJ | National Institutes of Health | |
Vector NTI | Thermo Fisher Scientific | https://www.thermofisher.cn/cn/zh/home/brands/invitrogen.html |
OVERVIEW OF THE EXPERIMENTAL DESIGN
STEP-BY-STEP PROCEDURE
Plasmid construction [ TIMING 1 week]
2 The primers used in this protocol |
Primer | Sequence |
SMT3-ATGL -F | GGCGAAGCTTGCATGTTCCCGAGGGAGACCAA |
SMT3-ATGL -R | TTATAGCGGCCGCTCAGCAAGGCGGGAGGCCAG |
S47A -F | CACATCTACGGAGCCGCGGCAGGGGCG |
S47A -R | CGCGGCTCCGTAGATGTGAGTGGCGTTG |
S47D -F | CACATCTACGGAGCCGACGCAGGGGCGC |
S47D -R | GTCGGCTCCGTAGATGTGAGTGGCGTTG |
S87A -F | CCTCTGCATCCCGCGTTCAACCTGGT |
S87A -R | CGCGGGATGCAGAGGACCCAGGAACC |
S87D -F | CCTCTGCATCCCGACTTCAACCTGGT |
S87D -R | GTCGGGATGCAGAGGACCCAGGAACC |
T101A -F | TGTCTACTAAAGGCGCTGCCTGCTGA |
T101A -R | CGCCTTTAGTAGACAGCCACGGATG |
T101D -F | TGTCTACTAAAGGACCTGCCTGCTGA |
T101D -F | GTCCTTTAGTAGACAGCCACGGATG |
T210A -F | CGCGTCACCAACGCGAGCATCCAGTT |
T210A -R | CGCGTTGGTGACGCGAAGCTCGTGGA |
T210D -F | CGCGTCACCAACGACAGCATCCAGTT |
T210D -R | GTCGTTGGTGACGCGAAGCTCGTGGA |
T372A -F | ATGAAAGAGCAGGCGGGTAGCATCT |
T372A -R | CGCCTGCTCTTTCATCCACCGGATA |
T372D -F | ATGAAAGAGCAGGACGGTAGCATCT |
T372D -R | GTCCTGCTCTTTCATCCACCGGATA |
S393A -F | GACCATCTGCCTGCGAGACTGTCTGA |
S393A -R | CGCAGGCAGATGGTCACCCAATTTC |
S393D -F | GACCATCTGCCTGACAGACTGTCTGA |
S393D -R | GTCAGGCAGATGGTCACCCAATTTC |
Y378A -F | AGCATCTGCCAGGCGCTGGTGATGA |
Y378A -R | CGCCTGGCAGATGCTACCCGTCTGCT |
Y378D -F | AGCATCTGCCAGGACCTGGTGATGA |
Y378D -R | GTCCTGGCAGATGCTACCCGTCTGCT |
S396A -F | CCTTCCAGACTGGCGGAGCAGGTGGA |
S396A -R | CGCCAGTCTGGAAGGCAGATGGTCA |
S396D -F | CCTTCCAGACTGGACGAGCAGGTGGA |
S396D -R | GTCCAGTCTGGAAGGCAGATGGTCA |
S406A -F | CTGCGACGTGCCCAGGCGCTGCCCTCTG |
S406A -R | CGCCTGGGCACGTCGCAGTTCCACCTGC |
S406D -F | CTGCGACGTGCCCAGGACCTGCCCTCTG |
S406D -R | GTCCTGGGCACGTCGCAGTTCCACCTGC |
S430A -F | GTACGAAACAACCTCGCGCTGGGGGACG |
S430A -R | CGCGAGGTTGTTTCGTACCCAGTTGGGT |
S430D -F | GTACGAAACAACCTCGACCTGGGGGACG |
S430D -R | GTCGAGGTTGTTTCGTACCCAGTTGGGT |
3 Reagents of PCR reaction |
Component | Volume |
5× EasyPfu buffer | 10 µL |
2.5 mmol/L dNTPs | 6 µL |
10 µmol/L forward primer | 1 µL |
10 µmol/L reverse primer | 1 µL |
Template DNA | 1.5 µL |
Nuclease-free water | 30 µL |
EasyPfu polymerase | 0.5 µL |
Total | 50 µL |
4 Procedures for amplification PCR |
Step | Temperature | Time |
Initial denaturation | 94 °C | 5 min |
25–35 Cycles | 94 °C 45–72 °C 72 °C | 40 s 40 s 60 s per kb |
Final extension | 72 °C | 10 min |
Hold | 4 °C | Forever |
5 Reagents of digestion reaction |
Component | Volume |
10× buffer | 5 µL |
PCR product/plasmid | 40 µL/1 µL diluted in 39 µL Nuclease-free water |
Restriction enzyme 1 | 2.5 µL |
Restriction enzyme 2 | 2.5 µL |
Total | 50 µL |
6 Reagents of ligation reaction |
Component | Volume |
10× T4 ligation buffer | 2 µL |
PCR product | 15 µL |
Plasmid | 2 µL |
T4 ligase | 1 µL |
Total | 20 µL |
7 Reagents of site-directed mutation |
Component | Volume |
5× Phusion HF buffer | 10 µL |
10 mmol/L dNTPs | 1 µL |
10 µmol/L forward primer | 2.5 µL |
10 µmol/L reverse primer | 2.5 µL |
DMSO | 1.5 µL |
Template DNA | 1.5 µL |
Nuclease-free water | 30.5 µL |
Phusion DNA polymerase | 0.5 µL |
Total | 50 µL |
8 Procedures for site-directed mutation |
Step | Temperature | Time |
Initial denaturation | 98 °C | 30 s |
25–35 cycles | 98 °C 45–72 °C 72 °C | 5–10 s 10–30 s 15–30 s per kb |
Final extension | 72 °C | 5–10 min |
Hold | 4 °C | Forever |
Protein expression [ TIMING 2–3 days]
1 The yield of recombinant wild-type ATGL and mutants assessed by SDS-PAGE. A Protein expression of wild-type ATGL and mutants induced by IPTG. Wild-type ATGL, S47D, S87A and S87D (a), S47A and S87A (b), S430D and SMT3 (c), S430A and S430D (d) were expressed in bacteria cells. B Bacterial lysates of wild-type ATGL and mutants used for the TAG hydrolase assay. The content of ATGL and mutants was standardized by scanning the density of protein bands. See also the supplementary Fig. S1 |
Adiposome preparation [ TIMING 2–3 h]
TAG hydrolase assay [ TIMING 4 h]
3 The expected outcome of the TAG hydrolase assay. A Scheme of ATGL binding adiposomes that are radiolabeled by triolein [9,10-3H(N)] and catalysing TAG hydrolysis. The radiolabeled TAG was catalyzed to release radiolabeled oleic acid. B The enzymatic activities of wild-type ATGL and mutants measured using adiposome platforms. Data represent mean ± s.e.m., n = 3. ∗p < 0.05, ∗∗p < 0.01, two-tailed t-test. This figure is reprinted by permission from Elsevier ref. (Maet al. 2021) |
ANTICIPATED RESULTS
DISCUSSION
9 Comparison of ATGL activity assays |
Substrate | Label | Radiation | Time | Reference |
Lipid emulsion | 3H-triolein | 40,000 cpm/nmol triolein | 3 h | (Schweigeret al. 2008; Zimmermannet al. 2004) |
Micelle | 3H-triolein | 117,660,000 cpm/nmol triolein | 3 h | (Duncanet al. 2008) |
Purified LDs | 3H-oleate | 1,660 cpm/nmol TAG | 3 d | (Schweigeret al. 2008) |
Adiposome | 3H-triolein | 752 cpm/nmol TAG | 4 h | (Maet al. 2021) |
NBD-TAG vesicle | NBD-TAG | 8,000 fluorescence units/nmol TAG | 8 h | (Rajanet al. 2021) |