Recent
years have witnessed the emergence of bacterial semiorganelle
encapsulins as promising platforms for bio-nanotechnology. To advance
the development of encapsulins as nanoplatforms, a functional and
structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel
for an enzyme cargo in its cavity, which could be replaced with a
foreign cargo, resulting in a modified encapsulin. Here, we characterize
the native structure of B. linens encapsulins with
both native and foreign cargo using cryo-electron microscopy (cryo-EM).
Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality
of the encapsulin for an in vitro surface-immobilized
catalysis in a cascade pathway with an additional enzyme, glucose
oxidase. We also demonstrate the in vivo functionality
of the encapsulin for cellular uptake using mammalian macrophages.
Unraveling both the structure and functionality of the encapsulins
allows transforming biological nanocompartments into functional systems.
A big
hurdle for the use of protein-based drugs is that they are
easily degraded by proteases in the human body. In an attempt to solve
this problem, we show the possibility to functionalize TM encapsulin
nanoparticles with an mEETI-II knottin miniprotein from the cysteine-stabilized
knot class. The resulting particles did not show aggregation and retained
part of their protease inhibitive function. This imposes a protection
toward protease, in this case, trypsin, degradation of the protein
cage. The used chemistry is easy to apply and thus suitable to protect
other protein systems from degradation. In addition, this proof of
principle opens up the use of other knottins or cysteine-stabilized
knots, which can be attached to protein cages to create a heterofunctionalized
protein nanocage. This allows specific targeting and tumor suppression
among other types of functionalization. Overall, this is a promising
strategy to protect a protein of interest which brings oral administration
of protein-based drugs one step closer.
Ro b i n Kl e m En c a p s u l i n s a s F u n c t i o n a l Pr o t e i n Na n o c a g e s En c a p s u l i n s a s F u n c t i o n a l Pr o t e i n Na n o c a g e s II
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