Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin

Abstract

The encapsulation of enzymes and other proteins within a proteinaceous shell has been observed in many bacteria and archaea, but the function and utility of many such compartments are enigmatic. Efforts to study these functions have been complicated by the size and complexity of traditional protein compartments. One potential system for investigating the effect of compartmentalization is encapsulin, a large and newly discovered class of protein shells that are typically composed of two proteins: a protomer that assembles into the icosahedral shell and a cargo protein packaged inside. Encapsulins are some of the simplest known protein shell systems and readily self-assemble in vivo. Systematic characterization of the effects of compartmentalization requires the ability to load a wide range of cargo proteins. Here, we demonstrate that foreign cargo can be loaded into the encapsulin from Thermotoga maritima both in vivo and in vitro by fusion of the cargo protein with a short C-terminal peptide present in the native cargo. To facilitate biochemical characterization, we also develop a simple and rapid purification protocol and demonstrate the thermal and pH stability of the shell. Efforts to study the biophysical effects of protein encapsulation have been problematic in complex compartments, but the simplicity of assembling and loading encapsulin makes it an ideal system for future experiments exploring the effects of encapsulation on proteins.

Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

“…This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins (Rurup et al, 2014; 2015; Cassidy-Amstutz et al, 2016). …”

Prokaryotic nanocompartments form synthetic organelles in a eukaryote

“…A large variety of native cargo proteins has been identified in bacteria and archaea, including peroxidases and ferritin-like proteins involved in stress response pathways 11,[14][15][16] . Using Escherichia coli as a host, it has been shown that packaging of non-native proteins into the encapsulins from Thermotoga maritima and Brevibacterium linens can be achieved by fusion of targeting peptides to the intended cargo 17,18 .…”

Encapsulin carrier proteins for enhanced expression of antimicrobial peptides

“…All HB‐Enc fusions were transformed and expressed in E. coli C43(DE3) cells from a P T7 promoter; this system was previously found to express highly soluble encapsulin cages (Cassidy‐Amstutz et al, ). All N‐terminal HB‐Enc constructs showed overexpression of soluble HB‐Enc protein by SDS‐PAGE and anti‐His 6 Western blot (Figure a).…”