Experiments
1. Construction of pQE-30-hGK2 recombinant plasmid:
(1) Material: human glucokinase sequence with BamHI and SalI
restriction enzyme cutting site and pQE-30 vector
(2) Digested the hGK2 sequence and pQE-30 vector with BamHI and SalI. Subject the digested products to agarose gel electrophoresis, cut the gel and extract the DNA fragments.
(3) Use T4 ligase for ligation at 22℃ for 1h, and take 2 μL of ligation product. The ligation product was transformed into the competent cells of E. coli M15 by the heat shock method. Add 1mL of antibiotic-free LB medium and culture at 37 ℃ for 1h. After centrifugation at 6000rpm for 2min, remove the supernatant, and the coat precipitate on LB solid medium containing Ampicillin and Chloramphenicol for overnight culture.
(4) The next day, pick up three colonies on LB medium plate and incubate each of them into 3mL LB medium containing ampicillin for overnight culture. Preserve the bacterial solution with glycerol, and extract the plasmids contained in the transformants with the Plasmid Extraction Kit.
(5) Use BamHI and SalI double enzyme digestion to identify, and sequence the positive plasmids.
(2) Digested the hGK2 sequence and pQE-30 vector with BamHI and SalI. Subject the digested products to agarose gel electrophoresis, cut the gel and extract the DNA fragments.
(3) Use T4 ligase for ligation at 22℃ for 1h, and take 2 μL of ligation product. The ligation product was transformed into the competent cells of E. coli M15 by the heat shock method. Add 1mL of antibiotic-free LB medium and culture at 37 ℃ for 1h. After centrifugation at 6000rpm for 2min, remove the supernatant, and the coat precipitate on LB solid medium containing Ampicillin and Chloramphenicol for overnight culture.
(4) The next day, pick up three colonies on LB medium plate and incubate each of them into 3mL LB medium containing ampicillin for overnight culture. Preserve the bacterial solution with glycerol, and extract the plasmids contained in the transformants with the Plasmid Extraction Kit.
(5) Use BamHI and SalI double enzyme digestion to identify, and sequence the positive plasmids.
2. Expression and purification of 6×his-hGK2 protein:
(1) Inoculate the positive strains to 20 mL of Ampicillin (100 μg/mL) and Chloramphenicol (50 μg/mL) in LB medium
overnight in a shaker at 37°C, 210 rpm. The next day, all the bacterial cultured medium was transferred into 1L of
LB medium containing Ampicillin (100 μg/mL) and Chloramphenicol (50 μg/mL) at 37°C 210 rpm until OD600 is around
0.6-0.8. Add IPTG until the final concentration is 0.3 mM, and the culture was continued to incubate at 25 ℃, 210
r/min for 20 h.
(2) Collect the bacteria cells at 4 ℃, 12000 rpm, centrifuge for 2 min, remove the supernatant and collect the precipitate.
(3) Add 30mL of binding buffer (20 mM Tris, 0.5 M NaCl, 10 mM Imidazole, pH = 8.0) and mix well. Then use an ultrasonic crusher to break the bacteria (4 ℃, 300W, sonicate for 10 s, stop for 5 s, a total of 30 min). collect the supernatant after centrifuging at 4℃, 12000 rpm for 40 min. During centrifugation, balance the Ni-NTA resin column with binding buffer, and then add the supernatant to the resin column and bound at 4 ℃ for 2 h.
(4) During this period, mix the protein several times to fully combine with the resin. Make the supernatant slowly pass through the resin column, and then remove the non-specific binding protein with 7.5mL binding buffer, 15mL elution buffer1 (20 mM Tris, 0.5 M NaCl, 20 mM Imidazole, pH = 8.0), and elute it with 7.5mL elution buffer2 (20 mM Tris, 0.5 M NaCl, 150 mM Imidazole, pH = 8.0).
(5) SDS-PAGE verification and stain it with Coomassie blue staining solution. Select the flow-through containing high-purity protein for dialysis.
(6) Load the purified protein into a 35 KDa dialysis bag and dialyze three times at 4°C for 5 h each time using dialysis buffer (25 mM HEPES, 25 mM KCl, 2 mM MgCl2, 1 mM DTT, pH = 7.1). Concentrate the protein to 2.24 mg / mL at 3000 g, 4°C using a 30 KDa concentration tube. After sub-packaging, store it at -80 ℃ for subsequent experiments.
(2) Collect the bacteria cells at 4 ℃, 12000 rpm, centrifuge for 2 min, remove the supernatant and collect the precipitate.
(3) Add 30mL of binding buffer (20 mM Tris, 0.5 M NaCl, 10 mM Imidazole, pH = 8.0) and mix well. Then use an ultrasonic crusher to break the bacteria (4 ℃, 300W, sonicate for 10 s, stop for 5 s, a total of 30 min). collect the supernatant after centrifuging at 4℃, 12000 rpm for 40 min. During centrifugation, balance the Ni-NTA resin column with binding buffer, and then add the supernatant to the resin column and bound at 4 ℃ for 2 h.
(4) During this period, mix the protein several times to fully combine with the resin. Make the supernatant slowly pass through the resin column, and then remove the non-specific binding protein with 7.5mL binding buffer, 15mL elution buffer1 (20 mM Tris, 0.5 M NaCl, 20 mM Imidazole, pH = 8.0), and elute it with 7.5mL elution buffer2 (20 mM Tris, 0.5 M NaCl, 150 mM Imidazole, pH = 8.0).
(5) SDS-PAGE verification and stain it with Coomassie blue staining solution. Select the flow-through containing high-purity protein for dialysis.
(6) Load the purified protein into a 35 KDa dialysis bag and dialyze three times at 4°C for 5 h each time using dialysis buffer (25 mM HEPES, 25 mM KCl, 2 mM MgCl2, 1 mM DTT, pH = 7.1). Concentrate the protein to 2.24 mg / mL at 3000 g, 4°C using a 30 KDa concentration tube. After sub-packaging, store it at -80 ℃ for subsequent experiments.
3. Screening of glucokinase agonists:
(1) The principle of this experiment is that GK catalyzes the phosphorylation reaction of ATP and glucose to
generate glucose-6-phosphate. Glucose-6-phosphate reacts with excess NAD+ to produce NADH under the action of
glucose-6-phosphate dehydrogenase. Therefore, the activity of GK can be reflected by measuring the absorbance
change of NADH at 340 nm.
(3) The reaction is initiated by adding 12 μL ATP using the Flexstation 3 Multifunction Microplate reader
workstation and immediately detecting the absorbance changes at 340 nM, reflecting the maximum reaction rate of GK
by the slope size.
(4) We validated the platform with the positive compound PF-04937319, and the EC50 of the positive compound is 0.93 μM.
(5) We used this platform to screen the compounds and found a small molecule 13926 with glucokinase activation activity. The EC50 was 87 nM and the maximum activation rate was 1.21.
(2) Based on this principle, we established a screening and
evaluation system for glucokinase agonists. The volume
of the reaction system is 120 μL. First, each component except the compound is dissolved into solution and then
added to a 96-well plate, then DMSO and the compound to be tested are added in the sequence. The content of each
component is as the following chart shows,
| Component | Volume (μL) | Final Concentration |
|---|---|---|
| Ultrapure Water | 51.6 | |
| Glucose | 12 | 5mM |
| G6PDH | 12 | 1mM |
| NAD | 12 | 5U/mL |
| 10×Buffer | 12 | 25mM HEPES, 25mM KCl, 2mM MgCl2,1mM DTT |
| BSA | 6 | 0.10% |
| LGK2 | 1.2 | 18.7μg/mL |
| Compound | 1.2 |
(4) We validated the platform with the positive compound PF-04937319, and the EC50 of the positive compound is 0.93 μM.
(5) We used this platform to screen the compounds and found a small molecule 13926 with glucokinase activation activity. The EC50 was 87 nM and the maximum activation rate was 1.21.
4. Detect the effect of active compounds on insulin secretion induced by glucose:
(1) Incubate 2×105 of Ins-832/13 cells in a 24-well cell culture plate with 500 μL medium which is placed in an
incubator with 5% CO2, 37°C overnight.
(2) The next day, aspirate the medium. Add 500μLof KRB buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 10 mM Hepes, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH = 7.3) with 2.8mM glucose. Starve the cells for 2h. And then replace the material with KRB buffer containing 5.5 mM glucose and 20 μM compound 13926. Incubate the cells for 2h.
(3) Supernatant of the absorbing medium was transferred to a 1.5 mL centrifuge tube at 600 rpm and centrifuged at 4℃ for 5 min. Take out the supernatant, and dilute it until the volume grew to 10 times the original. Test it with an insulin assay kit. After the cells were lysed at 100 μL RIPA, the protein concentration was determined as an internal reference using the BCA protein concentration determination kit.
(2) The next day, aspirate the medium. Add 500μLof KRB buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 10 mM Hepes, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH = 7.3) with 2.8mM glucose. Starve the cells for 2h. And then replace the material with KRB buffer containing 5.5 mM glucose and 20 μM compound 13926. Incubate the cells for 2h.
(3) Supernatant of the absorbing medium was transferred to a 1.5 mL centrifuge tube at 600 rpm and centrifuged at 4℃ for 5 min. Take out the supernatant, and dilute it until the volume grew to 10 times the original. Test it with an insulin assay kit. After the cells were lysed at 100 μL RIPA, the protein concentration was determined as an internal reference using the BCA protein concentration determination kit.
5. Detect the protective effect of active compounds on pancreatic islet cells:
(1) Ins-832 / 13 cells were incubated with a density of 5 × 105 / well. Incubate the cells in 96-well plates.
Culture them overnight in a 5% CO2, 37°C cell incubator.
(2) On the second day, add STZ with a final concentration of 0.4 mM and 13926 with a final concentration of 20 μM. Incubate them for 24 h.
(3) On the third day, suck out the medium, add the medium containing 0.5 mg / mL MTT, and continue to incubate for 4 h. After that, aspirate the medium. Add 100 μL DMSO to each hole, and shake the plate at 400 rpm for 10 min. Measure the absorbance at 490 nm with a spectrum max M5 multi-functional microplate reader.
(2) On the second day, add STZ with a final concentration of 0.4 mM and 13926 with a final concentration of 20 μM. Incubate them for 24 h.
(3) On the third day, suck out the medium, add the medium containing 0.5 mg / mL MTT, and continue to incubate for 4 h. After that, aspirate the medium. Add 100 μL DMSO to each hole, and shake the plate at 400 rpm for 10 min. Measure the absorbance at 490 nm with a spectrum max M5 multi-functional microplate reader.
6. Expected Results:
1. The recombinant plasmids pQE-30-hGK2 and pGEX-4t-1-hGK2
were successfully constructed.
2. Successfully purified 6×His-hGK2 protein and GST-hGK2 protein.
3. Establish an hGK2 agonist screening platform, evaluate the activities of the two proteins, and use the platform to screen the active compounds.
4. It is proved that the compound can enhance insulin secretion and protect pancreatic islet cells.
2. Successfully purified 6×His-hGK2 protein and GST-hGK2 protein.
3. Establish an hGK2 agonist screening platform, evaluate the activities of the two proteins, and use the platform to screen the active compounds.
4. It is proved that the compound can enhance insulin secretion and protect pancreatic islet cells.