FIG. 2.
The Pup C terminus is sufficient for pupylation. Ectopic expression of his6-pup or his6-pup91 (ΔN-term-Pup) and subsequent Ni-nitrilotriacetic acid (NTA) purification of pupylated proteins from M. smegmatis. (A) Coomassie brilliant blue (CBB)-stained SDS-PAGE gel of Ni-NTA purified pupylomes. (B) Immunoblot (IB) analysis of purified proteins using antibodies to His5. (C) Anti-Ino1 immunoblot shows Ino1 is pupylated with either full-length or truncated Pup. The pupylomes were purified from wild-type M. smegmatis cultures harvested at an optical density at 580 nm (OD580) of 1. The amount of protein loaded was normalized based on protein concentration of the eluted proteins, as determined by A280 using a NanoDrop spectrophotometer.