Part:BBa_K5121023

Reb1: RebB N-terminal Cys

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).

Figure 1. R body mechanism in nature as illustrated by Polka and colleagues (2016). The unique ability of R bodies to burst endosomes makes them attractive candidates for nano-based drug delivery.

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 cysteine maleimide conjugation at the N-terminus of rebB — facilitating the attachment of hundreds of thousands of drug molecules down the length of assembled R bodies. See the design page for more information.

Cysteine maleimide conjugation is a form of Michael addition, in which the thiol of the cysteine acts as a nucleophile to react with maleimide, forming a thiosuccinimide adduct (Figure 2). Through this reaction, drugs with maleimide groups can hence be reacted onto proteins with readily accessible cysteines.

Figure 2. Cysteine maleimide conjugation reaction mechanism. This conjugation reaction can be used to attach hundreds of thousands of drug molecules down the length of assembled R bodies.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 806
  Illegal NheI site found at 868
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 748
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

As described in the design section, a two-fragment PCR was run on the purified Reb1 plasmid to add the N-terminal CGGGGS motif to rebB. 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 3A,B). 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.

Figure 3. Cloning of RebB N-terminal Cysteines. PCR reactions were performed in duplicate or triplicate. A) Short fragment PCR products amplifying the insert. B) Long fragment PCR products amplifying the backbone.

Successful expression and purification of R bodies was first confirmed by imaging the R bodies in resuspension buffer (Figure 4A). The pH of the resuspension buffer was 7.5, in which we expect the R-bodies to remain in a coiled conformation like their wild-type counterpart (BBa_K5121011). This was observed in our microscopy images of these samples. We next tested the extension behaviour of these modified R-bodies in pH 5 HCl (Figure 4B). Unfortunately, these modified samples did not extend in acid, unlike their wild-type R-body counterparts (Figure 4C). Further testing of the extension kinetics of this construct is required, since we have only tested a small set of conditions.

Figure 4. Purification and extension testing of RebB N-term Cys R bodies. A) Purified RebB N-term Cys R bodies R bodies in resuspension buffer (25 mM Tris pH 7.5, 100 mM NaCl) observed at 63x under phase contrast. B) RebB N-term Cys R bodies in pH = 5 HCl observed at 63x under phase contrast. These R-bodies remained contracted, despite the acidic conditions. C) Wild-type R-bodies extending in pH = 5 HCl for comparison.

Despite the inability of these R-body constructs to extend in pH 5 HCl, we attempted a cysteine-maleimide conjugation reaction with the dye sulfo-Cy5 to assess the accessibility of our modification. Interestingly, this conjugation appears successful, since the R-body pellets are stained blue after washing in MQW (Figure 5A). Further validation of this conjugation can be performed with fluorescence microscopy. The aim of characterising conjugation strategies on R-bodies is to use these proteins as a drug delivery system. In line with this aim, we next attempted conjugation of these constructs with aldoxorubicin (Figure 5B). This conjugation reaction was successful.

Figure 5. Thiol-maleimide conjugation of sulfo-Cy5 maleimide and aldoxorubicin. A) Cys-N RebB (left) compared to a Reb1 control (right) after conjugation. Both samples have been washed thrice with MQW. B) Aldoxorubicin conjugates to Cys-N RebB forming a bright red pellet.

We then assessed the ability of these conjugated constructs to be endocytosed by EXPI293 cells (Figure 6). Cells showed a high endocytic propensity for aldoxorubicin conjugates and appeared to take them up in a vesicle-dependent manner (Figure 6B). Intriguingly, extensive cytoplasmic diffusion was observed in most cells (most notable in Figures 6B and 6C), a promising indicator that aldoxorubicin does not remain sequestered by the R bodies upon entering the cell.

Figure 6. Endocytosis of aldoxorubicin-Cys-N RebB conjugates in EXPI293 cells. A-C) DIC microscopy, fluorescence at 567 nm, and both modalities overlaid, respectively, of EXPI293 cells incubated with aldoxorubicin-Cys-N RebB. The R bodies were sterilised with 100% ethanol post-conjugation and then added to EXPI293 cell suspension culture. The cells were incubated at 37°C for 24h. Trypan blue was added in 1:1 ratio to an aliquot of the cell culture to quench extracellular fluorescence. A distinctly vesicular pattern (yellow square in B) overlaid with extensive cytoplasmic diffusion in most live cells is observed. The white arrow indicates a clump of contracted aldoxorubicin-N’ RebB bodies. D) An aliquot of aldoxoubicin-Cys-N RebB.

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.