Part:BBa_K2686001
Encapsulin protein
This is a BioBrick containing the sequence for Thermotoga maritima encapsulin from David Savage (Addgene #86405), a bacterial protein nanocompartment which self assembles to form a 60-mer.
Usage and Biology
Encapsulins are versatile proteins found in a variety of different bacteria (Giessen and Silver, 2017). In the case of this specific part derived from Thermotoga maritima, it can be used among other things to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids or the protein's C terminus. A cargo protein can also be loaded by fusing it with a tag binding to Encapsulin's interior surface (Cassidy-Amstutz 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 441 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 375
Illegal SapI.rc site found at 406
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.
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 SDS PAGE, where a band around 30.71kDa band is expected to form. Additionally due to Encapsulin's exceptional heat stability the 1.98MDa complex also appears on the gel after SDS denaturation.
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_K2686001 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_K2686001 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_K2686001. 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
1. Expression In order to observe whether BBa_K2686001-encoded encapsulin monomers were successfully expressed, we analysed our cell pellet using SDS PAGE. The pellet obtained from the 50 mL cultures was then resuspended in Tris Buffer Saline at an OD600 of 10. Once resuspended, the sample was cell lysed using sonication. Following sonication, the sample were span to separate the soluble and insoluble fragments form the whole cell lysate. 50 μL from each sample were obtained and stained with Laemmli reagent. We proceeded on with purification of the soluble fragment using column chromatography containing Strep-Tactin resin. The process involved packing
the column, equilibrating the resin and loading the soluble sample. Then a washing step was performed to remove any potential non bound nonspecific proteins. Then we eluted using competitive elution by loading BXT which competed with the T. maritima encapsulin monomers for binding sites with the resin, thus detaching the protein of interest from the column. Finally, we recycled the column ready for future purifications. From each of the samples obtained during the procedure we obtained 50 μL to use for SDS PAGE. According to our expression tests, this part was not suitable for successful production and purification of 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. 37ºC and 18ºC). 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, T. maritima encapsulin monomers (~ 32 kDa), which were produced in vivo by E. coli BL21(DE3), were concentrated in the insoluble fraction of BBa_K2686001- expressing bacteria even when the temperature of post-induction incubation was decreased from 37ºC (Figure 1A) to 18ºC (Figures 1B) to favour protein expression.
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_K2686001-expressing bacteria, we observed that, at both temperatures of post-induction incubation (i.e. 37ºC and 18ºC), 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_K2686001-encoded T. maritima encapsulin monomers at both production temperatures. Thus, DLS confirmed that, as it was observed from the SDS-PAGE gels, BBa_K2686001-encoded encapsulin monomers were mostly insoluble in in vivo protein expression systems and unable to self- assemble into T. maritima encapsulins.
Conclusion
We hypothesised that there may be some problems in T. maritima encapsulin in vivo isolation when applying the heat purification method for which BBa_K2686001 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_K3111102. which encodes T. maritima encapsulin monomers with an inserted StrepII tag that allowed for in vivo expression and purification using affinity chromatography.
References
Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.
Giessen, T. and Silver, P. (2017). Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nature Microbiology, 2, p.17029.
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