Team:UCL/Improve

Introduction Experiments

Improve: T.maritima Encapsulin with Strep Tag

Improved Part from the UCL iGEM 2019 team

Introduction

Thermotoga maritima encapsulins have previously been used by EPFL's 2018 iGEM team as protein cage nanocarriers for the development of a cancer vaccine. The EPFL 2018 iGEM team designed and expressed BioBricks encoding for the encapsulins with (BBa_K2686002) and without (BBa_K2686001) an internal HexaHistidine insert (4). The HexaHistidine sequence enhances the encapsulin's stability at higher temperatures and allows them to be heat purified following in vitro production (4).

We originally considered two commonly used encapsulin shells, one derived from Myxococcus xanthus and one fromT. maritima. We used structural modellingto help inform our choice. While the modelling was in progress, we performed some experiments with EPFL's encapsulin parts and then worked to improve them. We found modifying them to allow affinity purification helped increase purified yield and as well as purity of the final product.

Experiments

Figure 1: SDS PAGE of CFPS and heat purification of BBa_K2686001 and BBa_K2686002. M: molecular marker, 1: Unpurified CFPS of BBa_K2686001; 2: Heat purification BBa_K2686001; 3: Unpurified CFPS of BBa_K2686002; 4: Heat purification of BBa_K2686002

BBa_K2686001 and BBa_K2686002 are BioBricks encoding for T. maritima encapsulin monomers without and with a heat stabilising His tag repectively. They were created by the EPFL's 2018 iGEM, which was able to express the proteins, show they self-assemble into 60-mer cages in vitro using a cell free protein expression system (CFPS) and isolate them applying heat purification.

Before introducing the planned improvements in the previously designed part, we replicated the cell-free synthesis and heat purification of the encapsulins after EFPL's team kindly provided us with last year's team's plasmids. We used their protocol and expressed both parts. Figure 1 shows that encapsulins were expressed (bands indicated by red arrows, the encapsulin with the 6-His insert, as expected, is slightly bigger), however, no overexpression was observed. Unexpectedly, the heat purification did not yield pure T. maritima encapsulin.

We noted that the applications of the BioBricks were limited by some features of its design. Firstly, we tested the expression of the BioBricks in E. coli BL21(DE3), and found that we could not successfully purify the encapsulins in vivo using heat purification. In vivo encapsulin production was hindered by the aggregation of the protein monomers at different production temperatures (i.e. 37ºC and 18ºC). This was evident from SDS PAGE of the insoluble, soluble and heat-purified fractions obtained from our cell lysates. Figure 2 shows the in vivo produced encapsulin monomers (~ 32 kDa) were concentrated in the insoluble fraction even when the temperature of post-induction incubation was decreased from 37ºC (Figure 2A) to 18ºC (Figures 2B) to favour protein expression.

Figure 2: SDS PAGE of soluble (S), insoluble (I) and purified (P) fractions of BBa_K2686001 and BBa_K2686002 expressed in vivo at 37ºC (A) and 18ºC (B). Unlike in vitro production, the BioBricks were not present in the soluble fraction of the cell lysate and, consequently, they could not be heat-purified (evident by the absence of 32 kDa bands in the soluble and purified fractions). Nevertheless, a band of approximately 32 kDa was observed in the insoluble fractions indicated by the red rectangles. M= Ladder.

We found by doing dynamic light scattering (DLS) of BBa_K2686001 and BBa_K2686002 expressed in vivo, that due to their low-level of solubility they could not assemble to form 60-mer shells in vivo. T. maritima T=1 encapsulin monomers self-assemble into shells with a diameter of approximately 20-24 nm. Nevertheless, we observed that, at both incubation temperatures (i.e. 37ºC and 18ºC), no signal was obtained for molecules of that size. Instead, as displayed in Figures 3 and 4, DLS showed only the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm). Thus, DLS confirmed that, as it was observed from the SDS PAGE gels, the monomers encoded by those parts were mostly insoluble in vivo and unable to self-assemble into shells.

Figure 3: DLS of the heat-purified fractions obtained from cell lysates of E. coli BL21(DE3) expressing T. maritima encapsulin monomers without the HexaHistidine tag (4). The bacterial cultures were incubated at a post-induction temperature of 37ºC (A) and 18ºC (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.
Figure 4: DLS of the heat-purified fractions obtained from cell lysates of E. coli BL21(DE3) expressing BBa_K2686002 incubated at 37ºC (A) and 18ºC (B). The peaks revealed the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm) of the protein. The absence of a signal at 20-24 nm indicated that there were no assembled 60-mers encapsulin shells present.

We considered there may be several issues with heat purification. First, other bacterial proteins may be stable at the temperature at which the heat-purification is performed and, subsequently, could co-purify with the encapsulins. In fact, we observed that this was the case, as in the SDS PAGE gel we detected non specific bands (Figure 2), revealing additional proteins are present in the purified sample. This unspecific protein co-purification would not only reduce the purity of our target protein but may even contribute to lowering the solubility of the encapsulins(Figures 2-4).

Therefore, to address challenges these issues, we improved the existing BioBricks by introducing a StrepII tag-coding sequence that would enable the encoded encapsulins to be purified using affinity chromatography. We tested the efficacy of the StrepII tag in our improved BioBricks (BBa_K3111102 and BBa_K3111103). We were particularly interested in the tag's improvement to BBa_K2686002 because we wanted to conserve the HexaHistidine linker (GGGGGGHHHHHHGGGGG) between residues 43 and 44. This linker had been shown to convey exceptional heat stability and better hydrodynamic properties for the encapsulin multimer (4), properties that seemed attractive in manufacturing as the encapsulins could endure harsher processing conditions.

After growing the improved parts in the same conditions as EPFL's parts, we performed SDS PAGE and observed protein solubility was visibly improved after the addition of the StrepII tag (Figure 5). When we expressed BBa_K3111102 (encapsulin with a StrepII-tag without HexaHistidine insert) at 37°C, no encapsulin monomers were present in the soluble fraction (Figure 5A). However, decreasing the temperature to 18° yielded purifiable monomer in the soluble fraction of the cell lysate (Figure 5B).

Figure 5: SDS PAGE of soluble (S) and insoluble (I) fractions and affinity-purified (E1-E6) elutions of the improved HexaHistidine-lacking T. maritima encapsulin monomers (4). The improved part encoded an encapsulin monomer with an inserted StrepII tag which allowed the successful purification of the protein from the soluble fraction of the cell lysate after applying Strep-tag chromatography. Unlike its previously designed counterpart, our improved BioBrick (BBa_K3111102) could be expressed in vivo and was present in the soluble fraction obtained from E. coli BL21(DE3) cultures when these were incubated at a post-induction temperature or 18ºC (B). This was indicated by the presence of a band of approximately 32 kDa in the soluble, insoluble and the first elutions of the purified fraction samples that were run in the SDS PAGE gel (red rectangle, 4B). However, our protein construct was still insoluble when it was expressed at 37ºC (A). This was evidenced by the absence of bands with the size of encapsulin monomers (32 kDa) in the soluble and purified fractions obtained from the E. coli BL21(DE3) cells (red rectangle, 4A). M= molecular marker, L= flow-through fraction obtained after loading the sample into the column and W= wash fraction.

As hypothesised, the construct coding for the T. maritima encapsulin monomer with the HexaHistidine linkers was less prone to insolubility (4). This was evidenced by the SDS PAGE performed in the insoluble, soluble and purified (E2-E4 in Figure 6) fractions of cell lysates obtained from cultures which had been incubated at a post-induction temperature of 37ºC. Therefore, although there was no need to further enhance the solubility of the monomers in this instance, the expression of the improved constructs could even be enhanced by incubating the E. coli BL21(DE3) cultures at a post-induction temperature which favours protein expression (i.e. 18ºC).

Figure 6: SDS PAGE of soluble (S) and insoluble (I) fractions and affinity-purified (E1-E6) elutions of the improved HexaHistidine-containing T. maritima encapsulin monomers (4). The improved part encoded an encapsulin monomer with an inserted StrepII tag which allowed the successful purification of the protein from the soluble fraction of the cell lysate after applying Strep-tag chromatography. Unlike the HexaHistidine-lacking improved part (see Figure 5), the HexaHistidine-containing improved BioBrick was abundantly present in the soluble fraction of E.coli BL21(DE3) which were incubated at a post-induction temperature of 37ºC (4). This was expected as the insertion of said HexaHistidine tag had previously been reported to enhance the monomers heat resistance and stability (4). The encapsulin monomers resulting from the improved BioBrick (BBa_K3111103) could be purified and subsequently detected in the elutions resulting from the StrepII chromatography purification. This was indicated by the presence of a band of approximately 32 kDa in the soluble and insoluble fractions as well as in elutions 1-3 of the purified fraction (red rectangle). M= molecular marker, L= flow-through fraction obtained after loading the sample into the column and W= fraction generated after adding binding buffer to the column immediately after the sample loading and L elution collection.

Finally, we also tested our improved BioBricks by DLS. Although the DLS revealed the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm), it also revealed a considerable amount of assembled encapsulin shells. This was detected by the presence of peaks at diameter ≈ 20 nm for both improved parts (see Figures 7 and 8). Given that the solubility of the monomers encoded by the HexaHistidine-lacking improved part was lower than that of its HexaHistidine-encoding counterpart, it was not surprising that the intensity of the peak at that diameter was lower in the former (4). This is because a lower proportion of soluble monomers in a purified fraction would reduce the possibility of monomeric self-assembly into encapsulating 60-mers.

Figure 7: . DLS of the improved HexaHistidine-lacking T. maritima encapsulin monomers produced in vivo (4). In agreement with the results from the SDS PAGE gel (which revealed the absence and presence of soluble encapsulin monomers at post-induction incubation temperatures of 37ºC and 18ºC respectively), it was observed that, cultures incubated at a post-induction temperature of 37ºC did not assemble to form 20-24 nm 60-mer encapsulin shells (6A). However, a low concentration of self-assembled encapsulin cages was detected by DLS in samples proceeding from cell lysates of cultures incubated at a post-induction temperature of 18ºC (6B, red arrow). This was evidenced by a signal which, despite peaking at ≈ 37 nm, comprised a range of signals for differently sized molecules. Given that some signal was present for 20-24 nm diameter molecules, we concluded that the addition of a StrepII tag to the previously existing HexaHistidine-lacking T. maritima encapsulin monomer allowed its expression and self-assembly in vivo (4).
Figure 8: . DLS of the improved HexaHistidine-containing T. maritima encapsulin monomers produced in vivo (4). In agreement with the results from the SDS PAGE gel (which revealed the presence of soluble encapsulin monomers at post-induction incubation temperatures of 37ºC), it was observed that, 60-mer encapsulin monomers self-assemble into shells with a diameter of, approximately 20-24 nm (red arrow, red rectangle).

By the time we concluded our improvements to these two previously existing parts, our structural modelling analysis had already demonstrated T. maritima encapsulins would be a better candidate to develop our therapeutic platform. Having characterised the solubility and potential for self-assembly of the monomers encoded by both improved constructs, we decided to use the HexaHistidine-coding one to encode for the DARPin-directed protein shell that would transport the therapeutic cargo to breast cancer cells (4). Nonetheless, before proceeding with any further experiments, a final experiment confirming the enhanced solubility and potential for self-assembly of our improved protein construct was performed. The purified sample was examined by transmission electron microscopy (TEM) to check the presence of assembled encapsulins. The images obtained (Figure 9) confirmed the results that we had obtained after the DLS experiments: unlike existing BioBricks, our improved encapsulin monomer-encoding parts could be expressed and self-assembled in vivo.

Figure 9: . TEM image of the assembled encapsulins that were purified from the soluble fraction of E. coli BL21(DE3) bacterial lysates applying StrepII-tag affinity chromatography. As expected, the estimated diameter of the encapsulin shells that were visualised in the TEM images was approximately 20-24 nm.

Integration of the results obtained from the SDS PAGE, DLS and TEM studies confirmed that the modifications that we had introduced into the previously designed BioBrick BBa_K2686002 had improved it by increasing (i) variety of expression systems for which it is suitable, (ii) the range and specificity of chromatographic methods that could be employed to purify its protein product and (iii) the solubility and self-assembly capacity of the T. maritima encapsulin monomers in in vivo systems.

References

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