Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that can be attached to substrate proteins in Actinobacteria. The modification process is defined as pupylation and is associated with proteasome-mediated protein degradation in mycobacteria and streptomycetes. Here, we report the pupylation of Streptomyces hygroscopicus 5008 in vitro. Each component of the Pup system was expressed in Escherichia coli and poly-Pup chains were observed by western blot analysis. Though only one potential Pup substrate (SHJG_3659) was identified using MS/MS, we verified this candidate and other predicted substrates by a reconstituted Pup system in E. coli. In addition, we discuss the depupylation activity of Dop (a Pup activation enzyme). The results presented here show that pupylation exists in S. hygroscopicus and that a reconstituted Pup system can function in vitro in a heterologous host. (C) 2016 Elsevier Inc. All rights reserved.