GK, as a glucose receptor in pancreatic islets β cells, is mainly responsible for promoting insulin release and biosynthesis under glucose stimulation. The glucokinase agonists have a significant hypoglycemic effect and can significantly improve pancreatic islets’ β Cell function. So it is promising to screen glucokinase agonists for clinical treatment with GK protein.
In the past iGEM teams, there is no very detailed and complete screen platform of glucokinase agonists. For this reason, our team provides some valuable information for future iGEM teams by developing an in vitro screen platform.
We submit information on the E. coli protein expression plasmid-backbone. If a future team is committed to expressing and purifying target proteins, the plasmid-backbone can be used. Moreover, with the purified hGK2 protein, we developed an in vitro glucokinase agonists screen platform for future research. In addition, we have also provided another plasmid backbone with a GST tag for protein purification. The results showed that compound 13926, which was screened out by our in vitro platform. If a future team is also committed to screening for more glucokinase agonists, they can start with this in vitro screen platform

pQE30-backbone is one of the E. coli expression vectors, which uses a T5 promoter to regulate the expression of exogenous genes that could be induced by IPTG. We inserted the hGK2 DNA fragment into the BamHI and SalI sites, and the 6×His tag is fused at the N-terminal of the protein and is used for protein purification. The vector is a low-copy-number plasmid. When expressed in the prokaryotic system, the Amp+ resistance can be used to screen the right colony. This plasmid backbone can be used to express different proteins in the future.

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.

pGEX-4T-1-backbone is an E. coli expression vector, which employs tac promoter to regulate the expression of exogenous genes. In order to increase protein solubility, we inserted the hGK2 DNA fragment into the BamHI and SalI sites, and the GST tag is fused at the N-terminal of the protein, and the tag could be removed by thrombin. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Amp+ resistance can be used to screen the right colony. These plasmid backbones we submitted could be used to express different proteins in the future.