Difference between revisions of "Part:BBa K5121027"

Biology

Refractile bodies, known as R bodies, are ribbon-like protein complexes produced by certain strains of bacteria. Five classes of R bodies have been described — this part specifically encodes a modified type 51 R body containing four genes; rebA, rebB, rebC, and rebD. rebA and rebB constitute the primary structural components of R bodies, while rebC is thought to aid in the polymerisation process — the function of rebD remains unknown (Heruth et al., 1994). Under basic conditions, R bodies exist in a coiled-up conformation, but will extend in a telescopic fashion under acidic conditions (Heruth et al., 1994). In nature, R bodies are produced by bacterial endosymbionts of some Paramecia. Also referred to as kappa particles, these bacteria constitute the genus Caedibacter (Beier et al., 2002). These bacterial endosymbionts confer a killer trait to host paramecia — when released and taken up by sensitive paramecia, the bacteria are exposed to an acidifying environment in the endosome (Figure 1). These conditions cause the extension of R bodies inside the bacteria, rupturing them and the endosome to release a toxin to kill the host cell (Pond et al., 1989).

Part overview

Their ability to burst endosomes make R bodies appealing candidates for use in drug delivery, as they could hold the key to solving the endosomal escape problem. This composite part encodes an R body compatible with sortase A-catalysed conjugation at the C terminus of rebA — facilitating the attachment of hundreds of thousands of drug molecules down the length of assembled R bodies.

Sortase A is a bacterial transpeptidase enzyme that ligates C-terminal LPXTG motifs with N-terminal polyglycine motifs (Theile et al., 2013). Sortase A first recognises the C-terminal LPXTG motif, before cleaving the threonine-glycine peptide bond and forming a thioacyl intermediate with its active site cysteine residue. This intermediate can then be resolved via nucleophilic attack by an N-terminal polyglycine, resulting in an R1-LPXT(G)n-R2 linkage (Figure 2).

As such, to make this part sortase A-compatible, it contains an added LPETG motif at the C terminus of rebA. See the design page for more details.

Sequence and Features

Characterisation

As described in the design section, overlap extension PCR was run on the purified Reb1 plasmid to add the C-terminal LPETG motif to rebA. Two separate PCR reactions were performed with extension times dependent on the length of each fragment. The products of each PCR reaction were run on an agarose gel to determine if the PCR was successful (Figure 3). After verifying the PCR, the remaining products were run on a new gel, from which the products were purified.

A Gibson assembly was prepared with the purified PCR products. The Gibson assembly products were immediately transformed into DH5-alpha E. coli cells for propagation and BL21 E. coli cells for expression. Successful Gibson assembly was indicated by the growth of colonies on plates with kanamycin. Plasmids from selected colonies were extracted and sequenced to verify they contained the correct modified rebA sequence.

Successful expression and purification of R bodies was first confirmed by imaging the R bodies in resuspension buffer. Although the sample was extremely concentrated, which made it challenging to make out details, ring structures characteristic of R bodies could be seen amongst masses (Figure 5).

Further imaging was required to verify the presence of R bodies, and to confirm whether modified R bodies could still extend under acidic conditions. Indeed this confirmed the presence of R bodies, as extension was observed under pH = 2 (Figure 6A). Extension under pH = 5 conditions was also verified, although took longer (Figure 6B) — suggesting the motif perhaps impacts the kinetics of the extension mechanism.

To characterise conjugation onto this part, mNeonGreen was used to easily verify conjugation. mNeonGreen was expressed using a pCDFDuet-1_His_mNeon_TmTP plasmid. To make mNeonGreen compatible with sortase A-catalysed conjugation, an N-terminal triglycine motif was added, preceded by a TEV protease recognition sequence to cleave off the N-terminal methionine residue. This was added using the same method as with the LPETG motif on Reb1, using primers with complementary overhangs at the mNeonGreen N-terminus, along with two additional primers with complementary overhangs targeted at either side of an unwanted signal peptide at the C-terminus of mNeonGreen (Figure 7). The modified GGG-mNeonGreen was expressed, followed by nickel-affinity purification, overnight TEV cleavage, and size exclusion chromatography. The combination of these purification techniques yielded a stock of GGG-mNeonGreen to be used for conjugation to rebA via a sortase reaction.

Several variants of the sortase enzyme are commercially available. We selected a sortase pentamutant,which was engineered via directed evolution to have 140-fold enhancements in ligation activity (Chen et al., 2011). This sortase mutant is translationally fused to an N-terminal polyhistidine tag. This tag was used to perform Ni-NTA affinity chromatography on lysates of cells overexpressing the enzyme. Lysates were incubated with the resin for 3 hours in a binding buffer (50mM Tris pH 7.5, 300mM NaCl, 10mM imidazole). The unbound lysate was collected as a flow-through sample. The resin was washed 3 times with a washing buffer (50mM Tris pH 7.5, 300mM NaCl, 20mM imidazole). Finally, bound proteins were eluted multiple times using an elution buffer (50mM Tris pH 7.5, 300mM NaCl, 500mM imidazole). Aliquots were spared at each stage and run on an SDS-PAGE gel (Figure 8A). The eluted samples were combined and dialysed overnight with the buffer for size exclusion chromatography (25 mM tris pH 7.5, 150mM NaCl). Before size exclusion chromatography (SEC), the sample was concentrated to a volume less than 5mL, suitable for input into the SEC column. The absorbance at 280 nm for each 1mL SEC fraction was measured. Fractions with peaks at the expected size of sortase were collected and run in an SDS-PAGE gel to confirm the presence of sortase A (Figure 8B). SEC visibly removed contaminants enriched during the Ni-NTA chromatography. The pure sortase A samples were then combined and concentrated, forming our stock sortase solution for subsequent conjugation reactions.

To validate the activity of our enzyme before conjugations with our modified R-bodies, we performed a positive-control reaction. The two substrates were a domain of the chromatin remodeler CHD4 fused with an LPXTG motif and the modified GGG-mNeonGreen we developed. The reaction was incubated at room temperature overnight. The sortase enzyme, protein substrates, and reaction products were run on an SDS-PAGE gel. If conjugation is successful, a band shift to the combined mass of the two substrates is expected in the reaction lane (Figure 9).

Incubation of R bodies with sortase A and modified mNeonGreen led to successful conjugation of the fluorescent protein onto R bodies (see details of protocol here), as evident by the fact the pelleted R bodies were a bright green colour post-washing and the fact that R bodies observed under a microscope were also green (Figure 10). The bright green colour of the pelleted R bodies strongly suggested successful conjugation onto the R bodies, particularly given the lack of green observed in a sortase-free control sample (Figure 10A). Fluorescence microscopy of the conjugation sample verified the fluorescence was physically linked to the R bodies, confirming successful conjugation with mNeonGreen (Figure 10B).

To validate their use in drug delivery, modified R bodies must be readily endocytosed by cells. To test this, a 2 mL pellet of rebA-LPETG R bodies conjugated with mNeonGreen was resuspended and plated at 1:8 and 1:16 dilution into suspension EXPI293 cell culture, and incubated for 24 h or 48 h. Cells were later imaged by fluorescent microscopy and DIC, which have been overlain in Figure 11A-D. Incredibly, EXPI293 cells showed an impressive endocytic propensity for the fluorescent R bodies — being observed in almost all cells, with many cells containing multiple copies. Importantly cells appeared morphologically healthy — a key milestone in the characterisation of this part.

To assess endocytic efficiency, the average number of fluorescent R bodies per cell was determined at different dilutions and incubation times (Figure 11E). The mean number of R bodies per cell scaled with dilution factor — a highly promising dependence for therapeutic application. Interestingly, at higher concentrations (1:8), the number of R bodies per cell follows a bimodal distribution — cells are either very endocytic or not endocytic at all. This could potentially reflect the clumping behavior of R bodies, rendering cells that encounter R bodies unlikely to take up just one or two. Further investigation is required to better elucidate the cause and therapeutic implications of this heterogeneity.

References

Beier, C. L., Horn, M., Michel, R., Schweikert, M., Görtz, H.-D., & Wagner, M. (2002). The Genus Caedibacter Comprises Endosymbionts of Paramecium spp. Related to the Rickettsiales (Alphaproteobacteria) and to Francisella tularensis (Gammaproteobacteria). Applied and Environmental Microbiology, 68(12), 6043–6050.

Chen, I., Dorr, B. M., & Liu, D. R. (2011). A general strategy for the evolution of bond-forming enzymes using yeast display. Proceedings of the National Academy of Sciences - PNAS, 108(28), 11399–11404.

Heruth, D. P., Pond, F. R., Dilts, J. A., & Quackenbush, R. L. (1994). Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116. Journal of Bacteriology, 176(12), 3559–3567.

Polka, J. K., & Silver, P. A. (2016). A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo. ACS Synthetic Biology, 5(4), 303–311.

Pond, F. R., Gibson, I., Lalucat, J., & Quackenbush, R. L. (1989). R-body-producing bacteria. Microbiological Reviews, 53(1), 25–67.

Theile, C. S., Witte, M. D., Blom, A. E. M., Kundrat, L., Ploegh, H. L., & Guimaraes, C. P. (2013). Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nature Protocols, 8(9), 1800–1807.