Type 2 diabetes accounts for 90% of all kinds of diabetes caused by a declining response to insulin, the victims suffer from huge side effects. And the number of cases is increasing rapidly. Moreover, it is gradually spreading among younger crowds. Therefore, we find it helpful to many people to design a drug used to prevent symptoms of type 2 diabetes.
Glucokinase is a monomeric enzyme and serves as a “glucose-sensor” or “glucose receptor” in pancreatic cells and the liver, eliciting glucose-stimulated insulin secretion, and as glucose “gate-keeper” in hepatocytes, promoting hepatic glucose uptake and glycogen synthesis and storage. Due to its essential role in glucose homeostasis, it is important to develop a screening platform for GK activators. In this project, we chose pQE30 as a protein expression vector and purified the hGK2 protein in E. coli M15, and then the protein could be used in the screening platform.

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.

We have already collected the data and figures from our experiments. GK plays a role in transferring Glucose into Glucose 6-phosphate(G6P). hGK2, as a glucose receptor in pancreatic islets β cells, is mainly responsible for promoting insulin release and biosynthesis under glucose stimulation, as well as regulating pancreatic islets β cell survival and proliferation.
After transforming the hGK2 gene containing plasmid in the E. coli M13 strain, we detected the gene expression through SDS-PAGE. Because of the activity of the hGK2, we believe that if we can develop a glucokinase agonist compounds screen platform with hGK2 protein, it may be applied to clinical disease treatment, and improve the quality of life of patients.