We design the plasmid: The DNA fragment of the hGK2 was inserted into the BamHI and SalI sites of the pQE-30 vector (Figure 1).

In order to obtain the target fragments, we selected the appropriate endonuclease and digested both the DNA fragments and plasmid carrier simultaneously. We digested the DNA fragment hGK2 and plasmid pQE-30 with BamHI and SalI. Then we obtained the target DNA fragments (Figure 2) and ligated the fragments with T4 DNA ligase. Afterward, we transformed the recombinant plasmid into E. coli M15 competent cells and coated on the LB culture medium plate.

We inoculated 3 single colonies and extracted the plasmids. To verify the plasmids, we digested these plasmids with BamHI and SalI (Figure 3A). We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 3B), and the plasmid was successfully constructed. So far, we have successfully constructed the pQE30-hGK2 vector.

In Figure 3A, we can see that there are obvious bands of hGK2 and pQE30, proving that our recombinant cloning products were constructed successfully. In Figure 3B, we can see that there is no difference in the result of the template and construction, which represents the success of construction. This meant that we can carry out subsequent cell transfection and characterization qualitative detection.

In order to purify the protein, we cultured the M13 transformants in LB (Ampicillin/Chloramphenicol) and add IPTG to induce the protein expression when the OD600 reached 0.6-0.8. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used Ni-NTA column purification to purify the hGK2 protein. As shown in Figure 4, there are several clear bands which means the hGK2 protein was successfully expressed in the strain.

GK takes part in the first step of glycolysis, catalyzing glucose into Glucose 6-phosphate(G6P). We purified the hGK2 protein and developed an activity detection system using INS-832/13 cells to verify if our hGK2 works well.

We established a screening system for glucokinase agonists. The total volume of the reaction system is 120μL.

Finally, we used Flexstation 3 Multifunction microplate reader workstation to add 12 μL ATP to initiate the reaction and immediately detect the change in absorbance at 340 nm. The Maximum reaction rate is reflected by the slope of the curve.

As we can learn from the result (Figure 5), compound 13926 was finally discovered (maximum agitation rate, 1.21; EC50, 87 nM), which with higher activation activity than PF-04937319 (EC50, 930 nM).

Incubate 2×105 of INS-832/13 cells in a 24-well cell culture plate with 500μL culture medium overnight. The next day, replace the medium with 500μL of 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 and cultured for 2h. Then replace the medium with KRB buffer containing 5.5mM glucose and 20μM compound 13926. Incubate the cells for 2h. The supernatant of the medium was transferred to a 1.5 mL centrifuge tube and centrifuged at 600 rpm, 4℃ for 5 min. Dilute the supernatant and test it with an insulin assay kit. After the cells were lysed at 100μL RIPA, the protein concentration was determined as an internal reference using the BCA protein concentration determination kit. hGK2 enzyme activity detection platform was established and a positive compound PF-04937319 was used to prove the activity of hGK2 (Figure 6A).

Ins-832/13 cells were incubated with a density of 5 × 105 for each well. Incubate the cells in a 96-well plate. Culture them overnight in a 5% CO2, 37°C cell incubator. 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. On the third day, discarded 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. Detected the absorbance at 490 nm with a spectrum max M5 multi-functional microplate reader (Figure 6B).
The results indicated that 13926 effectively promoted insulin secretion and protected it from STZ damage in Ins-832/13 cells.