|
Fig. 1 - Supplemental 1 PafA fused with HaloTag exhibits high activity across a wide temperature range. (A) Schematic representation of Halo-PafA and three Pup substrates (HB-Pup, TS-sPup, SBP-sPup). HB, TS, and SBP refer to 6×His and BCCP, twin-STII (Strep-tag II), and streptavidin binding peptide, respectively. (B) In vitro self-pupylation of Halo-PafA (1 μM) with HB-Pup, TS-sPup, or SBP-sPup (10 μM) at 37°C, 28°C, and 20°C for 30 min. (C) Polypupylated Halo-PafAs were observed as high molecular weight bands after 2 hr pupylation reaction with three Pup substrates (HB-Pup, TS-sPup, SBP-sPup) at 37°C. Arrows indicate the Halo-PafA band without pupylation.