Difference between revisions of "Part:BBa K5301000"
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<figcaption><center><b><small><i>Figure 1 four lanes from right to left: marker, colonies 1, 2, 3, and 4. | <figcaption><center><b><small><i>Figure 1 four lanes from right to left: marker, colonies 1, 2, 3, and 4. | ||
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| + | We proceeded with the expansion culture of the single colony. 0.2mM IPTG was added for induction, and the culture was incubated in a shaker at 16°C and 200 rpm for 16 hours. Verification was performed through SDS-PAGE (Figure 2). The two lanes on the far right are the added MSP1E3D1 samples, and the bands are very unclear. This suggests that the concentration of our samples may be too high. | ||
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| + | <figcaption><center><b><small><i>Figure 2 The leftmost lane represents the protein molecular weight standard marker, while the two rightmost lanes show the crudely extracted MSP1E3D1 after IPTG induction. | ||
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Revision as of 14:24, 30 September 2024
MSP1E3D1 is a genetically engineered protein, which mimics the function of ApoA-I.
Usage and Biology
Nanodisc technology is a widely applicable approach to render membrane proteins soluble in aqueous solutions in a native-like bilayer environment, where the membrane proteins remain stable and active. The Nanodisc is a non-covalent structure of phospholipid bilayer and membrane scaffold protein (MSP), a genetically engineered protein, which mimics the function of Apolipoprotein A-1 (ApoA-1). The first MSP, MSP1, was engineered with its sequence based on the sequence of A-1, but without the globular N-terminal domain of native A-1. The MSP1E3D1 variant of MSP1 differs from MSP1 in the following facets: (1) It deletes the first 11 amino acids in the Helix 1 portion of the original MSP1 sequence (which is known separately as MSP1D1). The MSP1D1 protein is an N-terminal histidine-tagged protein with a TEV protease cleavage site between the histidine-tag and the protein sequence. (2) It repeats the Helix 4 (H4), Helix 5 (H5) and Helix 6 (H6) sequences of the original MSP1 sequence between the parent Helix 6 (H6) and Helix 7 (H7) segments of MSP1D1.
Experimental Design and Results
Our ultimate goal is to successfully construct a well-functioning nanoplate, thus it is crucial to explore the construction process and identify suitable conditions for the nanoplate. Literature has already demonstrated that MSP1E3D1 can successfully construct nanoplates [1], therefore, we have decided to use MSP1E3D1 as our target protein to explore the nanoplate construction process suitable for our experimental conditions.
We found the pMSP1E3D1 plasmid on Addgene, which can express MSP1E3D1 in E. coli. After transferring the pMSP1E3D1 plasmid into E. coli BL21 (DE3) cells, we obtained single colonies. PCR was performed on the single colonies, and the results were verified by agarose gel electrophoresis (Figure 1). Among the four selected single colonies, colony 4 most successfully obtained the target band.
We proceeded with the expansion culture of the single colony. 0.2mM IPTG was added for induction, and the culture was incubated in a shaker at 16°C and 200 rpm for 16 hours. Verification was performed through SDS-PAGE (Figure 2). The two lanes on the far right are the added MSP1E3D1 samples, and the bands are very unclear. This suggests that the concentration of our samples may be too high.
Sequence and Features