Purification of Full-Length Arc and Minimal Arc Gag
Figure 1. A 15% SDS-PAGE gel of the Arc protein after purification with sucrose and cesium chloride gradients. The RMR002 protein ladder from genemark bio. was used. Lanes 5 and 6 are the purified fractions from the cesium Chloride gradient as seen around the 45kDa mark as expected, and Lanes 1 and 3 show the cell pellet and Lysate respectively. These samples were then used for transmission electron microscopy.
Purification was also done with a sucrose cushion with size exclusion chromatography afterwards. However concentrations were too low and the gel came up as negative. The chromatogram can be seen below in Figure 2.
Figure 2. Chromatogram of the Arc full Size exclusion chromatography purification on the superdex 75 column. Although fractions were taken from the experiment, the protein amount was too low to be seen on the gel.
Figure 3. A 15% SDS-PAGE gel of the purification of Arc minimal construct first using a sucrose cushion followed by dialysis in 1XPBS and Size Exclusion Chromatography. The confirmed band is at about 40kDa indicating that Arc minimal construct is likely dimerizing. From left to right Lanes 2-10 contain the elutions taken according to the histograms seen in Figure 2. In Lane 11 there is the sample that was dialyzed. Lane 12 contains the cell pellet and Lane 13 contains the crude fraction from the sucrose cushion.
Figure 4. Chromatogram of Arc minimal construct size exclusion chromatography purification on the superdex 75 purification column. Samples were taken at Fractions 7-10 (whereby the two peaks were isolated), 11-13, 19-20 and 23.
Transmission Electron Microscopy
Transmission electron microscopy (TEM) confirmed the presence of spherical nanoparticles in both the full-length M. musculus Arc protein and minimal Arc Gag protein samples. Even in the absence of RNA cargo, the Arc-derived PNCs were capable of self-assembly in vitro. In both samples, the average PNC diameter was approximately 30nm +/- 10nm.
Figure 5. TEM image (60,000X magnification) of negatively-stained self-assembled full-length Arc PNCs. Scale bar = 200um.
Minimal Arc Gag
Figure 6. TEM image (100,000X magnification) of negatively-stained self-assembled minimal Arc Gag PNCs. Scale bar = 100um.
TEM also confirmed the presence of spherical nanoparticles in the P22 protein sample (without added scaffolding proteins; SPs) and P22 + SP-Cas9 protein samples. In the absence of the scaffolding protein, P22 capsid proteins were capable of self-assembly but with a significant variation in size and stability (many collapsed structures were evident in the sample). The P22 + SP-Cas9 PNCs had a much more consistent architecture but had a diameter of 120 +/- 5nm, which is approximately twice that of typical P22 virus-like particles. The 1:1 capsid to cargo ratio may have caused overloading of the PNCs, which caused an increase in overall size.
P22 coat proteins
Figure 7. TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids in the absence of scaffolding proteins. Scale bar = 500um.
Figure 8. TEM image (40,000X magnification) of negatively-stained self-assembled P22 capsids with the SP-Cas9 cargo. Scale bar = 200um.
Figure 9. Characterization of P22 purified P22 proteome using mass spectroscopy. Samples extracted from SDS-PAGE gels and digested with Trypsin. The data from the Mass spectroscopy analysis was then run on the software Thermo proteome Discoverer 184.108.40.2062.
The figure above shows an alignment of the P22 sequence based against the spectroscopy data. Consensus between the Sequence and Mass spectroscopy data, is shown in the green and has a value of 68.56%. The average cover success is about 70% according to the biomolecular analysis facility at the University of Virginia School of Medicine. Therefore, our mass spectroscopy analysis was successful and can inform the P22 structure and chemical composition. More importantly it proves that from comparing to our peptide sequence data to this experimental data, we were able to express and purify P22 successfully.
Analytical Ultracentrifugation of Minimal Arc Gag
We attempted to characterize our purified Arc-min protein with the use of sedimentation velocity data gathered through analytical ultracentrifugation. We prepared samples from both Arc-min and Arc-full, but were only able to observe any sedimentation from Arc-min. This suggests that there were insufficient amounts of Arc-full protein to run on AUC. The data that we observed for Arc-min indicate that the protein monomers aggregated which can be observed in the aggregation boundary in Figure 10 ( top left, absorbance; top right, relative intensity).(Refer to our Experiments page for more details on AUC.)
Figure 10. Analytical ultracentrifugation sedimentation results and simulations. (top left) Converted relative light intensity sedimentation rates into absorbance to compare to simulations. (top right) AUC sedimentation rates of Arc-min (top-right) measured in intensity of light at 217 nm and spun at 30,000 rpm. (bottom left) Modelled expected sedimentation rates of an Arc-min monomer and (bottom right) hexamer. Simulated data is based on estimated molar masses and globular anisotropies, using identical experimental conditions used in the actual experiment.
Cell Culture Transfection with Minimal Arc Gag
In progress! Visit us at the iGEM Jamboree for more info.
Purification of P22 and SP-Cas9
Figure 11. Nickel Affinity Chromatography Purification of SPCas9 using a gravity flow column shown on a 12% SDS-PAGE gel. Protein ladder is the 10-240kDa prestained ladder from Biobasic. The protein can be seen at the 245kDa mark. Lanes right to left show the stages of purification from Cell pellet, Lysate, supernatant flow through, column washes, elutions and the regeneration of the column.
The Purification of SPCas9 using nickel affinity chromatography was successful in terms of isolating the protein. Although yield is low compared to the levels of protein seen in the wash samples and lysate, protein was isolated. Further purification of smaller proteins was done by using a centrifugation spin column with a molecular weight cutoff filter of 50kDa. Loss of yield is likely due to overloading the column with supernatant and not enough nickel resin on the column compared to supernatant volumes. No further purification methods were done. These samples were used for both transmission electron microscopy imaging and experiments done by the University of Calgary as found here.The cell expression and purification protocol used can be found here.
Figure 12. Purification of P22 capsid using 35% sucrose solution and ultracentrifugation. Samples are shown on a 15% SD-PAGE gel. Protein ladder is the 10-240kDa prestained ladder from Biobasic. From lane Right to left shows the stages of purification. Lane 2 and 3 show the initial cell pellet and supernatant respectfully, Lane 4 shows the proteins within the sucrose and lane 5 shows the resulting crude fraction of P22 which is seen at the point shown at the red arrow.
We were unable to continue the purification due to time constraints; however, we were able to isolate the protein in the crude fraction as seen in Lane 5. The sample was then dialyzed against 1XPBS to get rid of residual sucrose. The sample was used for the Transmission electron microscopy experiments and the experiments done by the University of Calgary iGEM team.
Collaboration with the University of Calgary iGEM Team
In progress! Visit us at the iGEM Jamboree for more info.