Part:BBa_K4889006
Plac-GST-MST2 (mutant)
For expressing GST-MST2 (1-308) (mutant) protein in Escherichia coli BL21. Its affinity-expressed GST tag can be used to purify MST2 (1-308) (mutant) proteins. The part include T7 promoter, lac operator, RBS, GST tag, MST2 (1-308) (mutant), 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. The binding site predicted by virtual docking was mutagenized by PCR to determine the mode of action of CX6258. Based on the pET28a-GST-MST2 1-308 plasmid, we designed the primer sequences corresponding to the three amino acids to be mutated, i.e. GCA instead of CTT for L34A, GCA instead of GAA for E36A, and GCA instead of GTA for V42A, and then performed overlap PCR.
b. We performed protein purification of mutant MST2 (MST2 3 mutant). The experimental design was similar to the previous purification of MST2 and STRN3. The clear single band in lane 7 shows that the GST-MST2 3 mutant protein was successfully expressed and purified.
c. After the MST2 3 mutant protein was purified, we tested the role of CX6258 between MST2-STRN3 and MST2 3 mutant-STRN3. The results of lanes 1-4 show that after the binding site of CX6258 to MST2 is mutated, CX6258 no longer affects the interaction between MST2 and STRN3 (two bands in both lanes 1 and 2); the unmutated MST2 protein can still be affected by CX6258 and separated from STRN3 (only one distinct band in lane 3). Lanes 5 and 6 were used to exclude the effect of non-specific binding of GST-MST2 to HST tag or GST tag to HST-STRN3 on the results. Lanes 7-11 were used as the INPUT of each component and were used as a control to make the results more convincing. Overall, this result validates that the sites where CX6258 interacts with MST2 are L34, E36, and V42, elucidating the mechanism by which this small molecule is able to disrupt the MST2-STRN3 interaction. In addition, the elucidation of this mechanism will increase the commercial and translational value of the project results.
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