Protecting Encapsulin Nanoparticles with Cysteine-Knot Miniproteins

Abstract

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.

Keywords: antibody substitute; bacterial nanocompartment; nanocage; protease inhibitor.

Figure 1

UV–vis spectrum of TM encapsulin with maleimide-coupled Oregon Green 488 dye attached to the exterior thiol groups.

Figure 2

Difference in trypsin activity when inhibited by (A) EETI-II, (B) mEETI-II, (C) RGD-knottin, and (D) trypsin inhibitor from bovine pancreas. The y-axis indicates the difference in absorbance at 405 nm compared with t = 0.

Figure 3

Inhibitory effects of TM-mEETI-II. (A) Rate of l-BApNA cleavage by trypsin in the presence of TM (circle), mEETI-II (triangle), and TM-mEETI-II (square). (B) TEM images of TM encapsulins with and without mEETI-II functionalization, before and after trypsin treatment. The scale bars represent 20 nm.