Part:BBa_K2683031
P22 Capsid
The P22 protein is originally from the P22 bacteriophage from Salmonella. The protein able to properly form capsids (made of 420 subunits) when the capsid interacts with a scaffolding protein. With successful interaction, it forms capsids spontaneously [1]. The P22 capsid protein has been previously used to encapsulate fluorescent proteins [2] and CRISPR Cas9 systems [3].
In order to highly characterize P22, we have used a series of experiments including protein purification and Transmission Electron Microscopy.
Figure 1-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 further experiments.
Figure 3- A transmission electron microscopy image of P22 capsid. Capsids formed with the presence of SPCas9 which we also purified. Capsids were found to be twice the expected size but has been seen in literature [2].
Figure 4- This is the mass spectroscopy data of the P22 capsid. Protein samples were digested with trypsin and run. The green highlighted parts and sequences show the consensus of our peptide sequence and the protein sample.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 177
Illegal AgeI site found at 783
Illegal AgeI site found at 1005 - 1000COMPATIBLE WITH RFC[1000]
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