Heterologous expression and purification of encapsulins in .

In recent years a large number of encapsulin nanocompartment-encoding operons have been identified in bacterial and archaeal genomes. Encapsulin-encoding genes and operons from GC-rich Gram-positive bacteria, particularly of the phylum Actinobacteria, are often difficult to overexpress and purify in a soluble form using standard expression systems Here, we present a protocol to heterologously overexpress encapsulin nanocompartments and encapsulin-containing operons in Successful encapsulin production begins with the transfer of a expression plasmid, encoding the gene(s) of interest, via conjugation to the model actinobacterium After growing the conjugated culture to the optimal optical density, protein production is induced by the addition of the inducer thiostrepton, followed by expression in liquid culture for 1-3 days. Cells are lysed and encapsulin proteins purified using ammonium sulfate precipitation and size exclusion chromatography. The method outlined in this protocol can be utilized to improve cargo loading and overall soluble expression of encapsulin systems when compared to expression in •Clone an encapsulin-encoding gene or operon into a expression vector.•Transfer the expression vector to via conjugation.•Heterologously express and purify empty or cargo-loaded encapsulins from