This part encodes a modified Thermotoga maritima Encapsulin protein BBa_K2686001 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).
Usage and Biology
The part can be used to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids as well as the protein's C terminus. Cargo proteins can also be loaded inside the nano-cage using a tag binding to Encapsulin's interior surface (Cassidy-Amstutz et al., 2016). The protein is modified with an additional amino acid sequence (GGGGGGHHHHHHGGGGG) between positions 43/44 granting it better stability and high heat resistance (Moon et al., 2014).
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 77 Illegal BglII site found at 492
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE 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 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 we used the protocol developed by the 2017 EPFL iGEM team Aptasense.
SDS PAGE of encapsulins expressed in cell free TX-TL system with Lysine-BODIPY fluorescent tRNA's (Imager settings used were for measuring fluorescein λex=494nm, λem=512nm ). The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes). Stars (✦) show lanes where 60-mers are present. From left to right: (L) Positive control with DNA coding for Luciferase (37kDa), (H) HexaHistidine Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes (✦) as well as the monomer around 31kDa, (R) Encapsulin (BBa_K2686001) without HexaHistidine linker also has a band for the 60-mer (✦), (N) Negative control (cell-free TX-TL expression without DNA and 100C denaturation), (Ladder) LC5928 BenchMark™ Fluorescent Protein Standard, (L) Positive control with DNA coding for Luciferase (37kDa), (H) HexaHis Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes (✦) as well as the monomer around 31kDa, (R) Encapsulin (BBa_K2686001) without HexaHistidine linker also showing the multimer (✦), (N) Negative control (cell-free TX-TL expression without DNA and 70C denaturation)
Assembly
The self assembly of the encapsulin 60-mer was first examined using SDS PAGE, where a high band is expected to form due to the high molecular weight and size of the 1.9MDa complex.
SDS PAGE of the different encapsulin proteins expressed by iGEM EPFL 2018. Before (B) heat purification, the pellet after heat purification (P) and the supernatant after heat purification (S). From left to right: 1-3 Negative control (cell-free TX-TL expression without DNA), 4-6 HexaHis Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, 7 HexaHis-OVA Encapsulin (BBa_K2686000) where bands are not easily discernible, 8 Ladder Precision Plus Protein™ All Blue Prestained Protein Standards #1610373, 9 HexaHis-OVA Encapsulin BBa_K2686000) where the monomer band is visible at 31kDa, 10-12 HexaHis Encapsulin (BBa_K2686002) where the bands for the 60-mer and monomer can be identified, 13-15 HexaHis-OVA Encapsulin (BBa_K2686000) where the bands can easily be discerned for both the monomer and 60-mer (note how the 60-mer band is more visible in the supernatant after heat purification)
DLS Measurements
Intensity
DLS measurement of Encapsulin BBa_K2686002 using a Zetasizer Nano ZS from Malvern Analytical determining the average particle size using signal intensity. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water. This plot shows a peak at 32.674nm.
Negative control, TX-TL cell free expression medium purified according to the same procedure described above. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water.
Counts
DLS measurement of Encapsulin BBa_K2686002 using a Zetasizer Nano ZS from Malvern Analytical determining the average particle size using the amount of counts. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water. Due to this there may be additional error sources. This plot shows a peak at 18.166nm
Negative control, TX-TL cell free expression medium purified according to the same procedure described above. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water. Due to this there may be additional error sources.
Volume
DLS measurement of Encapsulin BBa_K2686002 using a Zetasizer Nano ZS from Malvern Analytical determining the average particle size using volumes. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water. This plot shows a peak at 21.037nm which corresponds to the encapsulin protein cage within the literature (Putri et al., 2017; Moon et al. 2014).
Negative control, TX-TL cell free expression medium purified according to the same procedure described above. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water.
Conclusion
Since the intensity weighted distribution shows how well particles with different sizes are identified from a fit to the autocorrelation function of the scattering, any protein aggregates even when present in small numbers can greatly vary the result. As for the count and volume, they are converted from the intensity using the protein's refraction index and absorbance. Since these are not exactly known we had to use the presets for proteins and water, which introduces errors (Malvern user manual). By analyzing these three measurements we can determine that the actual size of our protein is likely to be in between these extremes (larger than 18.166nm and smaller than 32.674nm). There are different hypotheses, first of all that the DLS measurement by intensity is biased and that the counts and volume based sizes represent the true size, or alternately that the DLS intensity measurement is accurate and that the 180-mer of encapsulin is formed. Overall, these results suggest that the construct forms multimers.
References
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.
Putri, R., Allende-Ballestero, C., Luque, D., Klem, R., Rousou, K., Liu, A., Traulsen, C., Rurup, W., Koay, M., Castón, J. and Cornelissen, J. (2017). Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake. ACS Nano, 11(12), pp.12796-12804.
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).
