Part:BBa_K5121021
RebA Cysteine-Maleimide N-terminus
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. These conditions cause 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).
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 rebA — facilitating the attachment of hundreds of thousands of drug molecules down the length of assembled R bodies.
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. Through this reaction, drugs with maleimide groups can hence be reacted onto proteins with readily accessible cysteines.
Figure 1: The chemical reaction bonding the cysteine and the maleimide.
Design notes
Although in vivo, the reb genes are transcriptionally independent, here they are encoded in polycistronic fashion. Since wildtype rebA contains no cysteines (and neither does rebB), this composite part contains a modified version of rebA with an added N-terminal cysteine (BBa_K5121013). This modification was made using a two fragment PCR of the original reb1 plasmid (BBa_K5121011) with overlapping primers as described in the design notes for the basic part (BBa_K5121013).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 806
Illegal NheI site found at 868 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 748
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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