Difference between revisions of "Part:BBa K2686000"

Latest revision as of 03:27, 18 October 2018

Encapsulin with HexaHistidine insert and C-terminal OT1

This part encodes a modified Thermotoga maritima Encapsulin protein BBa_K2686001with an additional HexaHistidine (GGGGGGHHHHHHGGGGG) insert between amino acids 43 and 44 BBa_K2686002, 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).

The part was obtained from BBa_K2686005 by inserting a OT1 coding sequence.

Sequence and Features

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 λ_ex=494nm, λ_em=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)

SDS PAGE of the different encapsulin proteins expressed by iGEM EPFL 2018. Cell-free mixture Before (B) heat purification procedure outlined above, the pellet after the heat purification procedure (P) and the supernatant after the heat purification procedure (S). Triangles ▲ signify the location of encapsulin 60-mer bands to the right, whereas stars ✦ show encapsulin monomer bands to their right. From left to right: (1-3) Negative control (cell-free TX-TL expression without DNA), (4-6) HexaHistidine Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes ▲ as well as the monomer around 31kDa ✦. (7) HexaHistidine-OT1 Encapsulin (BBa_K2686000) where bands are not easily discernible, (8) Ladder Precision Plus Protein™ All Blue Prestained Protein Standards #1610373 , (9) HexaHistidine-OT1 Encapsulin (BBa_K2686000) where the monomer band is visible at 31kDa ✦, (10-12) HexaHistidine Encapsulin (BBa_K2686002) where the bands for the 60-mer ▲ and monomer can be identified✦. (13-15) HexaHistidine-OT1 Encapsulin (BBa_K2686000)

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.

Native PAGE gel of sfGFP, HexaHistidine-OT1 Encapsulin and HexaHistidine-OT1 Encapsulin sfGFP-tag. On the left is the fluorescent image of the gel and o the right is the gel after Coomassie staining. Before (B) heat purification procedure outlined in Purification section, Supernatant (S) after the heat purification procedure. Stars ★ show the bands for monomers of HexaHistidine-OT1 Encapsulin (BBa_K2686000,BBa_K2686006) with sfGFP-tag bound to them, increasing the molecular weight of the complex when compared to sfGFP alone. Triangles ▲ signify a 60-mer band to the right. From left to right: (1-2) Negative control (cell-free TX-TL expression without DNA), (2-4) HexaHistidine-OT1 Encapsulin (BBa_K2686000) shown by a ★. The difference in height between the bands of sfGFP compared to the ★ is striking and suggests that the sfGFP-tag binds to the HexaHistidine-OT1 Encapsulin monomers. In addition there seems to be a small amount of 60-mer indicated by ▲. (9-10) Encapsulin (BBa_K2686001) does assemble to form the 60-mer ▲ as seen on the stained gel. (11) HexaHistidine-OT1 Encapsulin and sfGFP-tag (BBa_K2686006), on the left the presumed ★ protein dimer is seen to be higher than the sfGFP in lane 12, no particular bands can be identified on the right. (12) sfGFP, fluorescent band is seen to be lower than for lane 11.

Mass Spectrometry

The Coomassie stained gel sent to the Proteomics Core Facility. The two bands that were used for mass spec are surrounded by black rectangles and are BBa_K2686002 (on the left) and BBa_K2686000 (on the right).

We sent the Coomassie stained gel to the Proteomics Core Facility.

The Proteomics Core Facility then washed the gel we provided, reduced and alkylated it, digested the proteins using trypsin and extracted the peptides to perform MALDI-TOF mass spec. The analysis of the results was also performed by the facility and we were provided the peptide sequence alignments onto the HexaHistidine Encapsulin-OT1 construct.

Alignment of peptides to HexaHistidine ENcapsulin-OT1

1% FDR cutoff

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.

Spectrum of FSIINFEKL at 1% FDR threshold.

Fragmentation table of FSIINFEKL at 1% FDR threshold.

95% probability cutoff

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% probability. 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.

Spectrum of FSIINFEK at 95% probability threshold.

Fragmentation table of FSIINFEK at 95% probability threshold.

Alignment of peptides to HexaHis Encapsulin

1% FDR cutoff

Here the HexaHistidine Encapsulin BBa_K2686002 sample's fragmentation results are aligned against the sequence of HexaHistidine Encapsulin-OT1 BBa_K2686000 using a peptide cutoff of 1% FDR. There are no peptides aligning to the SIINFEKL part which is the desired result since BBa_K2686002 does not have a OT1 peptide at its C terminus.

95% probability cutoff

Here the HexaHistidine Encapsulin BBa_K2686002 sample's fragmentation results are aligned against the sequence of HexaHistidine Encapsulin-OT1 BBa_K2686000 using a peptide cutoff of 95% probability cutoff. There are no peptides aligning to the SIINFEKL part which is the desired result since BBa_K2686002 does not have a OT1 peptide at its C terminus.

Conclusion

Our results demonstrate that we successfully incorporated the OT1 peptide at the C terminus of our encapsulin vaccine.

Dendritic Cell Uptake

Vaccine Validation

Two experiments were performed for the validation of our vaccine system. The first experiment tested the ability of dendritic cells to uptake encapsulin, our vaccine platform. The second experiment tested encapsulin’s ability to mediate the neoantigen presentation on MHC-I complexes on the surface of dendritic cells. Note that the word ‘encapsulin’, throughout the whole Vaccine Validation part refers to encapsulin with inserted Hexahistidine (HH) linker.

Validation of Encapsulin Uptake by Dendritic Cells

We hypothesized that an appropriate method of checking the ability of dendritic cells to uptake our vaccine platform encapsulin is through the fluorescent tagging the encapsulin protein, presentation of 60-mer nanoparticles assembled from encapsulin monomers to a dendritic cell culture, and then imaging to check for the vaccine uptake by the dendritic cells. For imaging, we used Nanolive’s precision tomographic microscope to acquire 3D spatial and olerlaid 2D fluorescent images of the uptake process.

Hexahistidine encapsulin was first expressed in our cell free expression system, that was supplemented with Promega’s lysine BODIPY, following this protocol (Lysine BODIPY Protocol). This way some of lysine residues present in our protein construct would be fluorescently labeled, making the the whole construct fluorescently labeled.

After expression and purification, the correct expression of Hexahistidine encapsulin and the correct incorporation of fluorescent lysines was checked by performing an SDS PAGE and fluorescently imaging the gel (Figure 1). This fluorescent image suggests that we have a fluorescent band just below the 32 kDa ladder band, for both HH encapsulin B and HH encapsulin S, but not in the negative control (N.C. in Fig. 1). This indicates the presence of encapsulin at its expected size of 31 kDa. The image also suggests the presence of fluorescent bands of high weight for HH encapsulin B and HH encapsulin S, but not N.C. These high bands refer to a very high molecular weight, which we believe indicates the presence of the encapsulin multimer (M.W. ~ 1.8 MDa). This gel image serves as a proof that we are able to express fluorescent HH encapsulin.

Murine bone marrow derived dendritic cells were obtained from the LBI lab at EPFL. These were cultured in Matrigel, presented with encapsulin, and imaged according to the following protocol (Mention protocol). In short, imaging was performed before encapsulin presentation, one hour after encapsulin presentation, four hours after encapsulin presentation, and six hours after encapsulin presentation and washing (six hours following encapsulin presentation, the dendritic cells were washed and imaged). Note that the Nanolive microscope is fitted with a miniature top stage incubator that keeps the cells at 37ºC whilst performing the imaging. 3D imaging was done using Nanolive’s Tomographic microscope. Fluorescence Imaging was done using FITC filter (Excitation maximum: 490 nm, Emission Maximum: 525 nm) on Nanolive’s tomographic microscope. We thank Nanolive for setting up the microscope and helping us in acquiring the images.

The iGEM team pre-processed the raw 3D tomographic and 2D fluorescent image files in the Demo version of Nanolive’s software STEVE. The processed files were exported from STEVE into the open source software Fiji, where a z projection of the 3D stack was acquired according to the max intensity. The output image was channel merged with the fluorescence channel output. Prior to that, contrast in the fluorescence channel was increased to attenuate background noise. The resulting figures are depicted in the following figure.

Nanolive Imaging outputs at multiple time points before and after encapsulin presentation.

Nanolive Imaging outputs at multiple time points before and after encapsulin presentation.

The images acquired before encapsulin presentation show dendritic cells in their immature round shape. Moreover, fluorescence imaging showed very faint fluorescence originating autofluorescence. One hour following encapsulin presentation, no significant change concerning the shape of the dendritic cells was observed. Fluorescence channels showed a diffuse fluorescence signal throughout the field, indicating the spread of fluorescent encapsulin throughout the mixture. Four hours post presentation, we could see that the dendritic cells had developed extensive processes indicating maturation of these cells. Fluorescence channels revealed localization of fluorescence particles within the processes and around the nuclei of the dendritic cells. We believe that these fluorescent particles indicate the presence of encapsulin aggregates in the processes and in endosomal areas in dendritic cells. Two hours later (6 hours post presentation and washing), the processes were more pronounced and the fluorescence was stronger, again revealing encapsulin aggregate localization to the processes and endosomal areas of the dendritic cells .

We also created a video of a 3D reconstruction of a dendritic cell using STEVE and Fuiji to represent the 3D shape of the dendritic cell following the encapsulin vaccine presentation. The video is presented below.

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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).