Difference between revisions of "Part:BBa K5301011"
| Line 15: | Line 15: | ||
===Induction and Purification=== | ===Induction and Purification=== | ||
<br> | <br> | ||
| − | + | <i>Induction Expression</i><br><br> | |
The spMSP1D1 plasmid was transformed into BL21(DE3) cells.Successfully transformed colonies were selected through colony PCR, and single colonies were picked for scaled-up cultivation. when the OD value reached 0.7, IPTG was added to a final concentration of 0.2 mM to induce expression. The cultures were shaken at 16°C, 200 rpm for 16 hours. After centrifugation, SDS-PAGE was used to verify the expression, and it was observed that spMSP1D1 was successfully expressed. (Figure 1)<br><br> | The spMSP1D1 plasmid was transformed into BL21(DE3) cells.Successfully transformed colonies were selected through colony PCR, and single colonies were picked for scaled-up cultivation. when the OD value reached 0.7, IPTG was added to a final concentration of 0.2 mM to induce expression. The cultures were shaken at 16°C, 200 rpm for 16 hours. After centrifugation, SDS-PAGE was used to verify the expression, and it was observed that spMSP1D1 was successfully expressed. (Figure 1)<br><br> | ||
Revision as of 14:37, 30 September 2024
spMSP1D1
Express the SpyCatcher-MSP1D1-SpyTag fusion protein to achieve self-cyclization of MSP1D1 for constructing nanodiscs, while the 6×His tag is used for subsequent protein purification.
Usage and Biology
Membrane Scaffold Protein 1D1 (MSP1D1) is a synthetic derivative of apolipoprotein A-I, which is a major component of human high-density lipoproteins. MSP1D1 is designed to self-assemble with synthetic phospholipids into discoidal nanoparticles known as nanodiscs. These nanodiscs are soluble and stable in aqueous solutions, preserving the native-like architecture of a phospholipid bilayer.They are used as a tool for studying membrane proteins and have applications in biotechnology and medicine.
However, in the production of nanodiscs, it is challenging to generate nanodiscs larger than 20 nm for the reconstitution of membrane protein complexes. In order to achieve the self-cyclization of MSP1D1 to produce circular nanodiscs (cNDs), we have fused SpyCatcher and SpyTag to the N-terminus and C-terminus of MSP1D1, respectively, to create spMSP1D1. The covalent isopeptide bond between SpyCatcher and SpyTag facilitates the self-cyclization of spMSP1D1. Compared to the methods of self-cyclization using sortase enzymes or split inteins, the introduction of the SpyCatcher-SpyTag system makes the production of spMSP1D1 more convenient and increases the yield accordingly [1].
The significance of this step in our project is to provide a theoretical basis and explore conditions for the subsequent cyclization of multimeric MSPs to construct nanodiscs. Furthermore, spMSP1D1 nanodiscs, once embedded with membrane proteins such as the Low-Density Lipoprotein Receptor (LDLR), will be used for validation in our cellular experiments.
Induction and Purification
Induction Expression
The spMSP1D1 plasmid was transformed into BL21(DE3) cells.Successfully transformed colonies were selected through colony PCR, and single colonies were picked for scaled-up cultivation. when the OD value reached 0.7, IPTG was added to a final concentration of 0.2 mM to induce expression. The cultures were shaken at 16°C, 200 rpm for 16 hours. After centrifugation, SDS-PAGE was used to verify the expression, and it was observed that spMSP1D1 was successfully expressed. (Figure 1)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 496
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 496
Illegal NheI site found at 64 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 496
Illegal BglII site found at 924
Illegal BamHI site found at 97
Illegal XhoI site found at 820 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 496
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 496
- 1000COMPATIBLE WITH RFC[1000]