Difference between revisions of "Part:BBa K2686002"

Revision as of 19:33, 21 October 2019

Encapsulin protein with HexaHistidine insert

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

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)

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.

In vivo Characterisation by Team UCL 2019

Scale-up Procedure

Day 1

Different batches of BL21(DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K2686002 sequence coding for the T. maritima T=1 encapsulin monomer. Transformed cells were grown in LB agar plates containing chloramphenicol and glucose. Plates were incubated at 37°C overnight.

Day 2

Transformed colonies containing pSB1C3 + BBa_K2686002 were used to prepare overnight starter cultures containing a total of 5 mL LB broth and chloramphenicol (5 μL). Cultures were incubated at 37 °C overnight.

Day 3

A 50 mL scale-up culture was prepared from a single starter culture containing cells carrying pSB1C3 + BBa_K2686002. The culture was incubated at 37°C until it reached an OD of 0.6. Once they reached OD 0.6, the cultures were induced by addition of 400 μΜ IPTG. The cultures were left to grow again overnight at 37 °C.

Day 4

The culture was collected and transferred into a 50 mL Falcon tube. It was spun for 10 minutes at 5000 rpm in order to pellet the cells. Then the supernatant was discarded, and the pellet frozen at -80 °C.

Expression Analysis

According to our expression tests, this part was not suitable for successful production and purification of HexaHistidine-containing T. maritima encapsulin monomers in E. coli BL21(DE3). In vivo encapsulin production was hindered by the aggregation of the protein monomers at different production temperatures (i.e. 37°C and 18°C). This was evidenced by the SDS-PAGE gels which we run to test the presence of T. maritima encapsulin monomers in the insoluble, soluble and heat-purified fractions obtained from our cell lysates. As shown in Figure 1, the HexaHistidine-containing T. maritima encapsulin monomers (~ 32 kDa), which were produced in vivo by E. coli BL21(DE3), were concentrated in the insoluble fraction of BBa_K2686002-expressing bacteria even when the temperature of post-induction incubation was decreased from 37°C (Figure 1A) to 18°C (Figures 1B) to favour protein expression.

Figure 1: Figure 1. SDS PAGE of soluble (S), insoluble (I) and heat-purified (P) fractions of the T. maritima encapsulin monomers expressed in vivo at 37°C (A) and 18°C (B) from the existing part BBa_K2686002. Unlike in CFPS production, this previously designed BioBrick was not expressed as soluble monomers in the soluble fraction of the cell lysate and, consequently, they could not be heat-purified. This was evidenced by the absence of bands with the size of encapsulin monomers (~35 kDa) in the soluble and purified fractions obtained from the E. coli BL21(DE3) cells. Nevertheless, a band of approximately 35 kDa was observed in the insoluble fractions of the cell lysates of cultures incubated at a post-induction temperature of 37°C and 18°C. The band size at which encapsulin monomers were present (insoluble fraction) or expected to be (soluble and purified fractions) is indicated by the red rectangles. M: PageRuler Plus Protein Ladder.

Self-Assembly

Due to the lacking or low-level solubility of T. maritima encapsulin monomers produced by in vivo expression of this part, these could not assemble to form the loadable 60-mer protein shells in in vivo systems. This was evidenced by dynamic light scattering (DLS). Under normal (i.e. soluble) conditions, T. maritima T=1 encapsulin monomers self-assemble into an encapsulin cage with a diameter of approximately 20-24 nm. Nevertheless, after performing DLS in the heat-purified soluble fractions obtained from BBa_K2686002-expressing bacteria, we observed that, at both temperatures of post-induction incubation (i.e. 37°C and 18°C), no signal was obtained for molecules ranging that size in the samples used. Instead, as displayed in Figure 2, DLS studies only detected the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm) of BBa_K2686002-encoded T. maritima encapsulin monomers at both production temperatures. Thus, DLS confirmed that, as it was observed from the SDS-PAGE gels, BBa_K2686002-encoded encapsulin monomers were mostly insoluble in in vivo protein expression systems and unable to self-assemble into T. maritima encapsulins.

Figure 3: Figure 3. DLS of the heat-purified fractions obtained from cell lysates of E. coli BL21(DE3) expressing T. maritima encapsulin monomers with the HexaHistidine tag. The bacterial cultures were incubated at a post-induction temperature of 37°C (A) and 18°C (B). The peaks revealed the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm) of the protein construct. The absence of a signal at 20-24 nm indicated that there were no assembled 60-mers encapsulin cages in the heat-purified fractions.

Conclusion

We hypothesised that there may be some problems in T. maritima encapsulin in vivo expression, resulting in low quantity of protein when applying the heat purification method for which BBa_K2686002 had originally been designed. Firstly, a few other bacterial proteins may be stable at the temperature at which the heat-purification was performed and, subsequently, co-purify with the encapsulins that were being targeted. In fact, we observed that this was the case in cell-based protein expression systems, as, in the SDS-PAGE gel, we detected bands indicating the presence of proteins with molecular weights different to 32 kDa (Figure 1). This revealed that not only the encapsulin monomers had been heat purified and, therefore, there were additional proteins present in the purified analysed sample. This unspecific protein co-purification would not only reduce the purity of our target protein in the purified soluble sample but could even contribute to lowering the solubility of the previously existing parts coding for encapsulin monomers (Figures 1 and 2).

To overcome the challenges that arose with the in vivo solubility and self-assembly capacity of the monomers encoded by this part, the UCL 2019 iGEM team re-evaluated the purification by expressing BBa_K2686002 with T7 promoter (BBa_J64997) and a strong RBS (BBa_K2306014). After showing that said features allowed protein expression in vivo, Team UCL 2019 designed BBa_K3111103. which encodes T. maritima encapsulin monomers with an inserted StrepII tag that allowed for in vivo expression and purification using affinity chromatography.

Improvement by iGEM Kyoto 2019

Fig.1 TmEncapsulin was purified more efficiently with Ni-NTA
TmEncapsulin expressed E.coli was sonicated lysed with sonication. In the “Lysate” lane, the lysate was loaded. In the “Heat” lane, heat-treated supernatant was loaded. In the “Ni-NTA” lane, the protein was purified with Ni-NTA beads from the lysate, then loaded.

Fig.2 Ni-NTA beads purification got about 5 times concentrated TmEncapsulin
SDS-PAGE that showed in Fig.1 was triplicated, then band intensities were quantified with ImageJ. Then each protein concentration of the solution was calculated.

Fig.3 Visual description of our second improvement
Different types of SpyCatcher fused protein can be displayed on SpyTmEnc

3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. CBB-stained.

We improved “Encapsulin protein with HexaHistidine insert” (BBa_K2686002)to “SPYtag inserted Tm Encapsulin” (BBa_K3185000). We introduced two modifications to the existing part. First, we improved purification efficiency by adding 6x-His tag to C-terminus. Second, we enabled displaying different types of proteins on the surface of protein capsule by inserting SpyTag.

1st improve point: Improve purification efficiency by adding 6x-His tag to C-terminus
TmEncapsulin is a protein found from Thermotoga maritima. TmEncapsulin assembles sphere-like protein capsule as 60mer. This protein capsule could be applied to different situations, and ways to overexpress with E-coli and purify have been researched for a long time.

Encapsulin on existing basic parts page promoted thermotolerance of native TmEncapsulin by introducing 6x-His tag between 43rd and 44th amino acid. After Expressing this by E.coli cell-free system, we can purify encapsulin by collecting supernatant after heat treatment and centrifugation.

We added 6x-His tag to the C-terminus of an existing part. This is due to make them available to protein purify by using Ni-NTA beads. However, in a paper suggested, Ni-NTA beads cannot bind to 6x-His tag added in C-terminus because it doesn’t display enough to the surface of the protein capsule [1]. Here, we placed HAtag between TmEncapsulin and 6x-His tag in expectation of C-terminus to display enough on the surface of the capsule. We compared E.coli lysate before purification with a heat-treated product and Ni-NTA purified product with SDS-PAGE.

Shown in the Fig.1 below, we succeeded in purifying Enacapuslin by using Ni-NTA beads. You can see that the product purified by Ni-NTA has a darker band. Also, we quantified band intensity, then calculated purified protein concentration. As shown in Fig.2, we were able to purify with five times as much concentration than the existing part by using Ni-NTA. This improvement means we can purify encapsulin more easily with high concentration.

2nd improve point: Protein display through SpyCatcher/SpyTag system

There are many possible usages of Encapsulin. One is protein displaying on the surface of the capsule. For example, iGEM EPFL 2018 which created the existing part also created Encapsulin with HexaHistidine insert and C-terminal OT1 (BBa_K2686000) which are antigens of “OT1”. This modification aims to display antigen to immune cells effectively.

In order to make protein displaying easier, we inserted SpyTag in TmEncapsulin. SpyTag forms an isopeptide bond with SpyCatcher when they are mixed [2]. In previous research about TmEncap, it is showed peptides inserted after 138th amino acid in TmEncap can be exposed on the protein capsule as a loop [1]. Furthermore, Bae et al. showed when SpyTag is inserted at the same position, SpyCatcher/SpyTag also forms a bond between SpyCatcher and SpyTag inserted TmEncap (SpyTmEnc) [3]. This modification enables us to display different types of protein on SpyTmEnc through SpyCatcher. (See Fig.3)

To demonstrate our SpyTmEnc can conjugate with SpyCatcher fused protein, we mixed SpyTmEncap with several kinds of protein. In lane 4 and 5, SpyTmEnc is loaded with or without SpyC. Only in lane 5, which is mixed with SpyC, the bigger band appeared. The molecular weight of each protein is SpyC: 15.37k, SpyTmEnc: 37.04k, so the conjugated band should be 52.41k. Therefore the bigger band looks objective conjugated protein. As a negative control, we also tested with an existing part: TmEncapsulin without SpyTag. Properly, TmEnc and SpyC did not show conjugated band as shown in lane 3. Also, as shown in lane 7 and 9, proteins fused with SpyCatcher are also conjugated to SpyTmEnc.

This result shows we succeed in conjugating several kinds of proteins to Encapsulin. This means that the arbitrary type of protein which fused with SpyCatcher can be displayed on the protein capsule.

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).

References(iGEM Kyoto 2019)

1 Moon, H., Lee, J., Min, J., and Kang, S. (2014). Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform. Biomacromolecules 15, 3794–3801.

2 Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., and Howarth, M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. U. S. A. 109.

3 Bae, Y., Kim, G.J., Kim, H., Park, S.G., Jung, H.S., and Kang, S. (2018). Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue. Biomacromolecules 19, 2896–2904.