Part:BBa_K1469003
glk
Glucokinase of Bacillus megaterium. The enzyme catalyses the phosphorylation reaction of glucose to glucose 6-phospahate.
Characterization by 2022 iGEM Team Nanjing_HS
A recombinant plasmid for expressing and purifying GK protein.
BBa_K4289008 pQE-30-GK
Contribution
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.
Engineering
How we design our plasmid
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).
How we build our plasmid
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.
Protein expression and verification
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.
Improvement
Our composite component, BBa_K4289008, is an enzyme that could be used to develop the screen glucokinase agonists platform.
As early as 2014, the iGEM14_Saarland team was committed to transferring glucose to glucose-6-phosphate and producing HA (BBa_K1469003). Based on their work, by reading literature and consulting experts in related fields, we found that glucokinase (hGK2) also could to screen glucokinase agonists in vitro. Therefore, we further optimized the glucokinase agonists screening platform and constructed a new composite part BBa_K4289008 to screen more kinds of glucokinase agonists that can be used to decrease blood sugar and promote islet β-cell survival.
In order to prove the function of our new composite part hGK2, we transferred the recombinant plasmid into E. coli M13 cells to express the hGK2 protein. We purified the protein and established an in vitro glucokinase agonists screening platform, and found compound 13926, which has an effect on decreasing blood sugar. Then, by culturing INS-832/13 cells with compound 13926, we detected the effect of compound 13926 in promoting islet β-cell survival. As a result, it was further confirmed that compound 13926 has the effect of decreasing blood sugar and promoting islet β-cell survival.
2. Results of Compounds Screening Using this Platform
（a）To Establish the screen system for glucokinase agonists
We established a screening system for glucokinase agonists. The total volume of the reaction system is 120μL. Component of the reaction system
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).
（b）Effects of 13926 on insulin secretion
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).
（c）Effects of 13926 on protecting effects of pancreatic islet cells
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.
Future Work
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.
Reference
1. Al-Hasani, H., Tschöp, M. H., & Cushman, S. W. (2003). Two birds with one stone: novel glucokinase activator stimulates glucose-induced pancreatic insulin secretion and augments hepatic glucose metabolism. Molecular interventions, 3(7), 367.
2. Wang, Z., Shi, X., Zhang, H., Yu, L., Cheng, Y., Zhang, H., & Duan, W. (2017). Discovery of cycloalkyl-fused N-thiazol-2-yl-benzamides as tissue non-specific glucokinase activators: design, synthesis, and biological evaluation. European Journal of Medicinal Chemistry,139, 128-152.
3. Zhang, J., Li, C., Shi, T., Chen, K., Shen, X., & Jiang, H. (2009). Lys169 of human glucokinase is a determinant for glucose phosphorylation: implication for the atomic mechanism of glucokinase catalysis. PLoS One, 4(7), e6304.
Sequence and Features
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