Part:BBa_K4889004
Plac-GST-MST2 1-308
For expressing GST-MST2 (1-308) protein in Escherichia coli BL21. Its affinity-expressed GST tag can be used to purify MST2 proteins. The part include T7 promoter, lac operator, RBS, GST tag, MST2 (1-308), and T7 terminator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1685
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 939
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 765
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1616
Illegal SapI.rc site found at 169
a.MST2, the key kinase upstream of the Hippo signaling pathway, contains two domains, kinase domain and SARAH. The kinase domain has kinase activity and its interaction with the CC domain of STRN3 has been demonstrated5. Therefore, we cloned MST2 1-308 (kinase domain, 921bp) into the pET28a-GST vector and expressed in fusion with the GST tag, which facilitated subsequent protein purification.
b. The plasmid was transformed into E. coli and bacterial PCR were used to measure the size of MST2 1-308. Originally 921 bp in size, MST2 was around 1500 bp due to the size of both ends. The outcomes demonstrated that the plasmid construction was effective.
c. The GST-MST2 1-308 protein is our target protein, and the "total" sample is a mixture of various proteins within the bacteria. To isolate our target proteins, we first processed the samples through steps such as centrifugation, which produced a "supernatant" and a "precipitate". These initial steps helped us separate soluble proteins (in the supernatant) from insoluble proteins (in the precipitate), allowing us to focus on the proteins we were interested in. The "flow" step plays a critical role in removing unwanted contaminants. We use a column to pass the sample through a stationary phase that has a high affinity for GST-MST2 1-308. This step effectively captures our protein of interest while removing other molecules. After "Flow", we performed "Wash 1" and "Wash 2". The purpose of these steps is to remove any remaining impurities and ensure that the GST-MST2 1-308 protein is as pure as possible. "Wash 1" and "Wash 2" help to reduce background noise during the final elution. Finally, we disrupted the binding between GST-MST2 1-308 and the stationary phase using reduced glutathione salt buffer (Elution) to obtain purified GST-MST2 1-308.
d. We use AlphaScreen technology in this part. We bind “Donor” and “Acceptor” beads with MST2 and STRN3 in our experiment, separately. Donor beads contain a photosensitizing agent (phthalocyanine) that, when irradiated at 680 nm, excites ambient oxygen to a singlet state. Excitation of each Donor bead generates approximately 60,000 oxygen singlets per second, resulting in a glowing of receptor beads. We used the public platform of the School of Life Sciences to perform the high throughput screening. The library of small molecules screened included TargetMol's Natural Compound Library, Approved Drug Screening Library, and Inhibitor Library, totaling 3106 molecules. MST2 and STRN3 binding tightness were determined by simultaneously measuring the fluorescence intensity of each well in a 384-well plate. The first two columns and the last two columns of the plate are negative and positive controls, respectively. For the negative control, we added only magnetic beads without protein, so the light intensity produced will be very low. And the light intensity produced by the positive control will be very high. Therefore, we can use this method to calculate the separation rate of MST2 and STRN3:
After the calculation, our group found CX6258 which has the highest separation rate for doing the rest of the experiment.
e. Functional validation of small molecule drug candidates using pull-down experiments Through the interview with Ph.D. Song during human practice, we learned that although the AlphaScreen technology has the advantages of a wide range of applications, including small molecules to large complexes, high speed, and high sensitivity, its main limitations should not be ignored. These include the sensitivity of the reaction system to bright light or prolonged room light; the trapping of singlet oxygen molecules by some compounds can reduce the optical signal; and the bleaching effect of donor beads makes single-pass signal detection preferable. To verify the screening results in the previous step, we performed pull-down experiments with CX6258 to clarify the separation of MST2-STRN3.
GST pull-down is a method to study protein interactions in vitro, and the basic principle is as follows: assuming that MST2 and STRN3 may have interactions, we will fuse MST2 with a GST tag. Then, GST-MST2, HST-STRN3, and Sephrose4B beads (which can specifically bind GST) are incubated for a certain period of time, the unbound proteins are washed sufficiently, the beads are boiled for SDS-PAGE electrophoresis, and finally radiolabeling is performed, and the corresponding bands of GST-MST2 and HST-STRN3 are seen, indicating that the two proteins are pulled down due to the interaction. If there is no interaction, only one band corresponds to GST-MST2.
The results showed that lane 1 was used to exclude the possibility of the GST tag interacting with STRN3. Lanes 4-7, as the INPUT of each component, served as a control and to make the results more convincing. Lanes 2 and 3 were used to test whether CX6258 could disrupt the MST2-STRN3 interaction. Lane 2, due to the addition of CX6258 in the sample, showed only one band in the SDS-PAGE (only GST-MST2 was pulled down), while lane 3 showed two bands (indicating that GST-MST2 and HST-STRN3 were pulled down, respectively), indicating that CX6258 has the ability to disrupt the MST2-STRN3 interaction.
f. A series of hit compounds can be obtained based on the AlphaScreen high-throughput screening system. To verify the reliability of this high-throughput screening system, we selected one of the candidates for the following in vitro pull-down and point mutation experiments to further confirm the accuracy of the screening results. From further human practice on the issue of future development of the drug, we learned that the current application of our drug in the treatment of hippo-related cancers belongs to the stage of lead compounds, and the subsequent chemical modifications need to be continuously carried out to achieve better efficacy and lower side effects. These optimizations are based on the understanding of the binding site of CX6258 with the target protein MST2 and the crystal structure of the binding site. Therefore, we used virtual docking prototypes to predict and validate the binding sites of both. Autodock Lab is a computer software widely used for molecular docking research of drug molecules. Its principle is based on the theories of molecular mechanics and computational chemistry, used to predict the binding ability and binding mode between compounds. By simulating intermolecular interactions, Autodock can provide important information about molecular binding and drug development for drug designers. We imported the protein sequence information of MST2 and the molecular structure information of CX6258 into the software, and obtained the following prediction result graph, showing that the binding sites are L34, E36, and V42 (steps for using Autodock virtual docking are shown in the protocol).
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