Difference between revisions of "Part:BBa K2686000"
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===Mass Spectrometry=== | ===Mass Spectrometry=== | ||
+ | ====Alignment of peptides to HexaHistidine ENcapsulin-OT1==== | ||
[[File:AlignmentOT1FDR.png|thumb|center|upright=3|This image was taken in Scaffold Viewer 4, where the peptides identified from mass spec are aligned to the HexaHistidine Encapsulin-OT1 sequence using a peptide cutoff threshold of 1% FDR. The OT1 peptide has alignments with peptides at the encapsulin’s C terminus which indicates that the OT1 peptide is successfully expressed.]] | [[File:AlignmentOT1FDR.png|thumb|center|upright=3|This image was taken in Scaffold Viewer 4, where the peptides identified from mass spec are aligned to the HexaHistidine Encapsulin-OT1 sequence using a peptide cutoff threshold of 1% FDR. The OT1 peptide has alignments with peptides at the encapsulin’s C terminus which indicates that the OT1 peptide is successfully expressed.]] | ||
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+ | [[File:(K)FSIINFEK(-)1%spectrum.PNG|thumb|center|upright=3|Fragmentation table of FSIINFEKL at 1% FDR threshold.]] | ||
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+ | [[File:PeptideFDR.png|thumb|center|upright=3|Fragmentation table of FSIINFEKL at 1% FDR threshold.]] | ||
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+ | [[File:AlignmentOT1Prob.png|thumb|center|upright=3|This image was taken in Scaffold Viewer 4, where the peptides identified from mass spec are aligned to the HexaHistidine Encapsulin-OT1 sequence using a peptide threshold of 95% FDR. The OT1 peptide excluding the last amino acid has alignments with peptides at the encapsulin’s C terminus which suggests that the OT1 peptide is successfully expressed.]] | ||
===Dendritic Cell Uptake=== | ===Dendritic Cell Uptake=== |
Revision as of 18:58, 17 October 2018
Encapsulin with HexaHistidine insert and C-terminal OT1
This part encodes a modified Thermotoga maritima Encapsulin protein derived from BBa_K2686005. The part is optimized for expression in E. coli and has an additional HexaHistidine (GGGGGGHHHHHHGGGGG) insert between amino acids 43 and 44, forming a loop on the interior surface of the encapsulin monomer providing higher heat resistance and stability, and better hydrodynamic properties (Moon et al., 2014). The C-terminus of the encapsulin is fused to a SIINFEKL (OT1) peptide which is displayed on the exterior surface of the encapsulin monomer as an antigen (Choi et al., 2016). SIINFEKL was chosen as it is a very popular model antigen sequence in research and a variety of antibodies targeting it are available, furthermore it is used as a tumor antigen model in scientific research and has been used in conjunction with encapsulin (Choi et al., 2016).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 77
Illegal BglII site found at 492 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 426
Illegal SapI.rc site found at 457
Characterization
A variety of different characterization techniques were used to assess the properties of the encapsulin protein cage.
Expression
A cell free T-expression system was used to synthesize the encapsulin proteins in vitro. The TX-TL cell free system is a robust way to express proteins (Sun et al., 2013), and was used by last year's EPFL iGEM team Aptasense.
Purification
After having tested a variety of purification procedures, heat purification at 70C for 20 minutes followed by cooling on ice for 15 minutes and a subsequent centrifugation at 12000g for 10 minutes was found to be the most efficient way of isolating the encapsulin (encapsulin ends up in supernatant).
Assembly
The self assembly of the encapsulin 60-mer was first examined using PAGE, where the monomer is seen around 32.9kDa as well as a high band due to the high molecular weight and size of the 1.98MDa complex, showing that the 60-mer is present.
Mass Spectrometry
Alignment of peptides to HexaHistidine ENcapsulin-OT1
Dendritic Cell Uptake
References
Choi, B., Moon, H., Hong, S., Shin, C., Do, Y., Ryu, S. and Kang, S. (2016). Effective Delivery of Antigen–Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection. ACS Nano, 10(8), pp.7339-7350.
Moon, H., Lee, J., Min, J. and Kang, S. (2014). Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules, 15(10), pp.3794-3801.
Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K. and Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), pp.751-755.
Sun, Z., Hayes, C., Shin, J., Caschera, F., Murray, R. and Noireaux, V. (2013). Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Journal of Visualized Experiments, (79).