Part:BBa_K5301011
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)
Figure 1 | SDS-PAGE analysis of the expression results of protein spMSP1D1. Lanes 1 was marker, lanes 4-6 were s crude extract of spMSP1D1.The gel was electrophoresed at 90V for 15 minutes, then at 150V for 45 minutes, stained with Coomassie Brilliant Blue, and subsequently subjected to protein gel analysis
Purification of spMSP1D1
We collect the bacterial culture and centrifuge to harvest the pellet.We resuspended the pellet in buffer A, sonicated to disrupt the cells, and centrifuged again. Using nickel bead affinity chromatography, we washed away impurities with a washing buffer and then eluted the protein using an elution buffer containing a gradient concentration of imidazole for purification, followed by SDS-PAGE analysis (Figure 2). It can be seen that a large amount of the target protein was purified in the 100 mM imidazole eluent and the 300 mM imidazole eluent; however, the purity was not high, with some non-specific bands present,which may be caused by dimerization or polymerization of spMSP1D1.Further purification by molecular sieve was required.
Figure 2 | SDS-PAGE analysis of the extraction results of protein spMSP1D1. Lanes 1-8 were marker, target protein, the eluents were obtained by using the imidazole concentration of 50, 100, 300, 500 mM elution buffer, protein effluent sample, washing buffer effluent sample, bead. The gel was electrophoresed at 90V for 15 minutes, then at 150V for 45 minutes, stained with Coomassie Brilliant Blue, and subsequently subjected to protein gel analysis.
Further purify spMSP1D1 using a molecular sieve
In order to obtain higher purity proteins, molecular sieve chromatography was used to purify proteins. After SDS-PAGE analysis (Figure 3), we obtained high purity spMSP1D1 and suspected spMSP1D1 dimer proteins, which were then concentrated and measured by ultrafiltration tube for subsequent preparation of nanodiscs.
Figure 3 | SDS-PAGE analysis of the product results of protein spMSP1D1. Lanes 1-3 were marker, possible protein spMSP1D1 dimer, protein spMSP1D1. The gel was electrophoresed at 90V for 15 minutes, then at 150V for 45 minutes, stained with Coomassie Brilliant Blue, and subsequently subjected to protein gel analysis.
Verify spMSP1D1 dimers or oligomers by Western Blot.
We performed a Western blot using the samples eluted with 300 mM imidazole and the putative dimeric samples purified by size exclusion chromatography. By utilizing anti-His antibodies, we aim to verify the presence of spMSP1D1 dimers and oligomers in the samples. (Figure 4)
Figure 4|Western blot result image
Functional Characterization
The construction and characterization of spMSP1D1 nanodiscs
We prepared nanodiscs with different lipid-to-protein ratios, and immediately performed native-PAGE characterization after the preparation was completed. Compared with lane 1, other lanes had a significant difference, which is similar to the band shape in the literature. We preliminary considered that it is successful.(Figure 5)
Figure 5|The first lane is a control for spMSP1D1 protein, and second lane is spMSP1D1 nanodiscs
We prepared transmission electron microscope samples using the spMSP1D1 nanodiscs and performed transmission electron microscopy imaging. The results are shown below (Figure 6). In the image, we can see that the black circular objects are the nanodiscs. Their diameter is approximately 10 nm, which is close to the theoretical value of 11nm. Surprisingly, we also observed the nanodiscs from the side.
Figure 6| Nagetive EM image of spMSP1D1 nanodiscs shows that the black circular objects in the image are the nanodiscs(a). We observed the nanodiscs from the side(b). According to the quantitative statistics of electron microscope results, the particle sizes of our nanodiscs are close to the value in the literature(c).
The construction and characterization of spMSP1D1 nanodiscs incorporating LDLR
We have constructed nanodiscs incorporating LDLR membrane proteins using spMSP1D1 at a defined ratio with lipids, following a similar method. The ratio of LDLR, spMSP1D1, and lipids was optimized to facilitate the reconstitution of membrane protein complexes within the nanodiscs.Based on the images from negative staining electron microscopy, we believe that spMSP1D1 nanodiscs incorporating membrane proteins have been successfully created.(Figure 7)
Figure 8|Nagetive EM results of nanodiscs incorporating membrane proteins
Reference
[1]Zhang, S., Ren, Q., Novick, S.J. et al. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nat Commun 12, 5451 (2021). https://doi.org/10.1038/s41467-021-25737-7
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]
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