Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Encapsulins as platforms for enzyme-based nanoreactors (#253)

Lachlan S.R. Adamson 1 , Yu Heng Lau 1
  1. School of Chemistry, University of Sydney, Sydney, NSW, Australia

The production of enzyme-loaded protein cages for use as engineered nanoreactors is an emerging area of synthetic biology. Current efforts to produce such nanoreactors are hampered by a lack of protein cages with native systems for loading cargo, instead relying on inefficient passive methods to load enzymes.1-2 Encapsulins are a recently discovered class of bacterial organelle composed of protein shells that naturally encapsulate protein cargo via self-assembly, providing a distinct advantage over other protein cages.3-4 However, there is currently a lack of fundamental understanding of the cargo-loading system and the effects of encapsulation on enzyme activity.

As part of a fundamental investigation, I expressed and purified a range of filled encapsulin particles from E. coli. Three variants of encapsulin, which assemble into particles of distinct sizes, were purified with and without protein cargo. Five cargo proteins, comprising of three enzymes and two haemoglobins, were purified inside encapsulin particles.

The effect of encapsulation on the stability and kinetics of two model enzymes, β-galactosidase and nitroreductase, was also investigated. Encapsulation appeared to enhance the thermal stability of β-galactosidase in a manner that was dependent on enzyme packaging efficiency. The maximum velocity and substrate affinity of β-galactosidase and nitroreductase were altered when encapsulated, in a manner that also appeared to be dependent on the packaging efficiency of the enzyme.

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