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Myxococcus Xanthus encapsulin protein

This gene encodes for the Myxococcus Xanthus encapsulin shell protomer EncA.

Sequence and Features

Usage and Biology

Research into bacterial metabolic compartmentalisation has led the discovery of a new class of bacterial proteinaceous structures named Bacterial Microcompartments (BMCs) in 1956, which act in an analogous fashion to eukaryotic organelles(1).

In the mid-1990s a new-class of structurally smaller and sequence dissimilar compartments called encapsulins within Brevibacterium Linens was discovered followed by the Pyrococcus furiosus and Thermotoga maritima encapsulins which revealed their capsid-like morphology loaded with cargo-proteins. To date more than 900 encapsulin systems with a diversity of putative cargo proteins have been identified spanning a remarkable breadth of microbial diversity and habitats(2).

High resolution crystallography has revealed that encapsulin nano-compartments are assembled entirely from a single protein protomer, making them distinct from the conventional eukaryotic organelles. The shell proteins are homologs of HK97 viral phages and assemble into defined icosahedral architectures of varying size depending on the species(3). They are classified into 2 classes determined from their triangulation number (T). T=1 encapsulins form smaller structures (20-24 nm in diameter) composed of 60 protomers while T=3 encapsulins are bigger (30-32 nm in diameter) and are composed of 180 monomers. Furthermore, they have multiple pores of 5–6 Å of varying chemical nature that control the exchange of small molecules between the cytosol and the lumen facilitating their metabolic function.

Research literature indicates 2 major roles of encapsulins: Bacterial protection against environmental stresses - especially oxidative and nitrosative stresses- by forming complexes with ferritin-like enzymatic proteins and hemerythrins(3). Participation in the global nitrogen cycle as they were identified within anammox bacteria. However, their presence in many less explored phyla like the Planctomycetes and Tectomicrobia might reveal other novel functions as this field moves forward.

Functionalisation

Team UCL 2019 has studied the expression and modularity of M. xanthus encapsulin. In BBa_K3111101, the encapsulin is flanked by a FLAG tag which allows chromatography column purification and immunostaining in order to observe the path of encapsulins whilst in contact with mammalian cells and the potential delivery pathway. We intended to evaluate the potential to use this encapsulin in terms of:

Variable cargo loading: Encapsulins’ natural ability to internalise cargo with high selectivity and affinity using a defined C-terminal targeting sequence allows their application in systems where non-native proteins need to be packaged.

Variable surface display: Fusion of additional genes on the C-terminus of the EncA would allow the surface displays of additional organic and proteinaceous moieties. In this case we are fusing DARPin_929 (add link which directs to that biobrick) in order to allow targeting to SK-BR-3 HER2 positive cells to test our initial hypothesis/ our potential drug delivery platform.

Experimental Results

DNA Analysis and Cloning

Figure 1: Test digest to confirm ligation of M. xanthus EncA gene in the pSB1C3 plasmid; a-d indicate repeats of different colonies containing the same plasmid cut with XhoI and XbaI type II restriction enzymes.

In order to confirm the ligation of the EncA gene encoding for the M. xanthus outer shell, we digested miniprepped plasmids from 4 different transformed DH5α colonies. As observed in figure 1, digestion with XbaI and XhoI indicated bands at the predicted sizes of 2166 and 892 bp (a band at 139 bp was expected; however its small size makes difficult to observe) for all 4 plasmid. This indicated that all 4 plasmids could be used to transform BL21* (DE3) to express the protein in bacterial cultures.

Day 1

Different batches of BL21 DE3 competent cells were transformed with empty pSB1C3 plasmids and pSB1C3 plasmids containing the EncA sequence coding for the M.xanthus capsid Transformed cells were grown in LB agar plates containing chloramphenicol and glucose. Plates were incubated at 37°C overnight.

Day 2

Transformed colonies for empty pSB1C3 and pSB1C3_EncA were used to prepare overnight starter cultures containing LB broth and chloramphenicol. Cultures were incubated at 37°C overnight.

Day 3

2 50 mL scale-up cultures were prepared from a single starter culture containing cells carrying pSB1C3 + BBa_K3111101. 1 50 mL culture was prepared from a single starter culture containing cells carrying empty pSB1C3. All cultures were incubated at 37°C until they 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 cultures were collected and transferred into 50 mL falcon tubes. Those were 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. The following growth curves were obtained: (might change labels accordingly)

While we would expect the control bacterial culture to grow at a faster rate compared to the induced culture after induction, this was not the curve as observed from the growth curves in Figure 2.

Protein Analysis

In order to observe whether the M. xanthus encapsulin shell was 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 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.

From Figure 3 it could be concluded that the encapsulin shell was successfully produced since the correct band at ⁓36 kDa is observed in both the soluble and insoluble fragment. It could also be concluded that encapsulins are mostly insoluble as we observe a much thicker band in the corresponding lane. (30-70% respectively).

Figure 3: SDS PAGE of EncA expression; L: PageRuler Protein Ladder S: Soluble Fragment I:Insoluble fragment.

Figure 4: SDS PAGE of EncA purification; M: PageRuler Protein Ladder, L: Load flowthrough, W: Wash, E2-4: Elution 2-4.

We proceeded on with purification of the soluble fragment using column chromatography containing an anti-FLAG 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 a 3X FLAG peptide which competed with the EncA-FLAg for binding sites with the resin, thus detaching the protein of interest from the column.

After executing the SDS PAGE we concluded 2 things. Firstly, we used most of the capacity of the column thus some of the EncA protein tagged with FLAG is not bound thus elutes during the loading step/ or some parts of the resin due to mishandling were inaccessible to the EncA thus elute. Secondly, we successfully eluted the EncA since similarly we obtain the bands at the appropriate position

Transmission Electron Microscopy

The purified samples were concentrated using Sigma Aldrich Centrifugal Filter with molecular weight cut of 10000 Da. The samples were spun for 15 min. Negative staining was performed in order to visualise the sample on Copper-carbon based grids.

The procedure was:

• Apply 5 uL of sample and let dry for around 2 minutes
  • After the 2 minutes the drop is drained not blotted
• On parafilm, pipette a drop of 2% uranyl acetate and distilled water.
  • First place the grid (on the sample face) on distilled water to remove excess sodium ions which could deposit
  • Drain the water
  • Place in uranyl acetate for 2 mins
  • Drain the uranyl acetate: This allows Negative staining to outline structure of the encapsulin
• Drain water, but not the blot in order to allow staining and outline of the structure.
• Then the samples are placed in a rod and placed in the TEM to visualise.

The following images at a relative magnification of around 150k were obtained:

Figure 5: Negative staining TEM images of E2 sample (Figure 4) obtained in FLAG-tag column; the red circle in b) indicates the presence of smaller variants of the assembled encapsulins.

From Figure 5, we can observe a concentrated sample of round-shaped assembled encapsulins. The majority of them lie in the range of 30 nm, however as indicated on fig. 5b there are smaller variants. This is probably explained due to self-assembly dynamics which ends up producing encapsulins composed of different number of monomer encapsulin proteins.

Dynamic Light Scattering

We wanted to further investigate the morphology and conformational changes of M. xanthus encapsulin under different physicochemical changes. Therefore we proceeded with Dynamic light scattering, a technique which uses light scattering fluctuation to determine the diffusion properties of particles in solution and conclude a range of hydrodynamic diameter of the particles present in the solution.

We started by determining the lowest concentration at which we could reliably measure the hydrodynamic diameter of our assembled encapsulins associated with FLAG tags.

Figure 6: Size Distribution of Encapculins by intensity (present within TBS); Initial concentration ∼ 1.3 mg/mL a) 1:10 dilution - Average size 39.79 nm, b)1:50 dilution - Average size 40.15 nm, c)1:100 dilution - Average size 39.29 nm.

We initially concluded that the threshold for measurements is 1:100 (around 0.01 mg/mL) since lower dilutions give wobbly results (dilution 1:200) From these 3 measurements we concluded that: the average diameter was 39.7 nm. We were expecting an average diameter of 33 nm, however we speculate that the FLAG tags surrounding the encapsulin must have increased the overall diameter. Moreover, the smaller variants more probably are included within the peak since the size difference is considerably small to generate a separate peak; also even if much smaller particles were present, the intensity is proportional to the diameter raised to the power of 6, therefore any peak would hardly be observed. Finally, we observed in all of the dilution a bigger species that was decreasing in size at greater dilutions. A possible reason could be that an aggregate was present due to the age of the sample. Through serial dilutions, resuspension could have possibly broken up the bigger aggregates into smaller ones.

Figure 7: Alteration of diameter after heating encapsulin sample for 10 min at 50 °C.

Figure 8: Alteration of diameter after addition of HCl to encapsulin sample to reach pH of ~ 1.

Figure 9: Alteration of diameter after addition of NaOH to encapsulin sample to reach pH of ~ 14.

After conducting these series of experiments we reach the following conclusions. From Figure 7 we can observe that an increase in temperature leads to both decrease and increase in diameter. These results could possibly be due to loss of stability making them smaller and consequent break down could lead to aggregation thus resulting in bigger diameter. The presence of a bell curve with peak at 28 nm means that also a proportion of encapsulins of 39 nm still exists. This is an indication of the high stability these structures possess even at elevated temperature. Exposing the encapsulins at very acidic or basic conditions leads to an increase in diameter as observed in both Figure 8 and Figure 9. Similarly, we think that this is associated with aggregation of broken up monomers into larger particles.

Conclusion

After analysis of results, we ultimately chose to go with T. maritima encapsulin (BBa_K3111003) as the basis of our drug delivery system, since modelling analysis showed lower possibility of steric hindrance issues as well as the fact that it only assemble into one conformation (N=60) unlike the M. xanthus encapsulin.

References

[1] Nichols RJ, Cassidy-Amstutz C, Chaijarasphong T, Savage DF. Encapsulins: molecular biology of the shell. Crit Rev Biochem Mol Biol. 3 de septiembre de 2017;52(5):583-94.

[2] Giessen TW, Silver PA. Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat Microbiol. 6 de marzo de 2017;2:17029.

[3] Giessen TW. Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science. Synth Biol Synth Biomol. 1 de octubre de 2016;34:1-10.