Part:BBa_K5301024
Multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MS
Usage and Biology
In order to produce large nanodiscs more conveniently, we hope to flexibly extend the length of MSP according to demand, and thus propose the concept of multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MSP fragments. We used NW15 as the basic MSP and selected three types of linkers (Spy/Sdy/Snoop) to achieve the connection of different MSP fragments through the formation of covalent bonds, and adopted rigorous design to prevent self-cyclization of each fragment of the multi-polymer MSP. Finally, the successful cyclization of large circular MSPs is characterized by the fluorescence of mCherry after the combination of mCherry [1-10] and mCherry [11].
We also utilized AlphaFold 2 to simulate the overall structure of multi-polymerized MSP, and obtained multi-polymerized MSP with the correct conformation, as shown in the figure.
Figure 1.Alpha Fold 2 prediction model of the overall structure of multi-polymerized MSP
Characterization
We attempted to mix the three components of multi-polymerized MSP in vitro under two different conditions: first, by centrifuging the bacterial cultures, resuspending them, mixing the three bacterial suspensions, and then sonicating the mixture, followed by incubating the protein mixture at 4°C; alternatively, by mixing the three highly purified proteins after purification through nickel affinity chromatography. We verified the efficiency of the assembly of the three components through SDS-PAGE, as shown in the figure below.
From the figure, it can be observed that compared to the monomeric MSPs, the mixed product exhibits two new bands at approximately 100 and 180 kDa. These are presumed to be multi-polymerized MSP.
Figure 2.SDS-PAGE analysis of the construction of multi-polymerized MSP.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3141
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 3141
Illegal NotI site found at 1720
Illegal NotI site found at 4038 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1239
Illegal BglII site found at 2323
Illegal BglII site found at 2695
Illegal BglII site found at 3557
Illegal BglII site found at 3929 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3141
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3141
Illegal AgeI site found at 1035
Illegal AgeI site found at 1828
Illegal AgeI site found at 2220
Illegal AgeI site found at 2980 - 1000COMPATIBLE WITH RFC[1000]
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