The production of SUMO-Bac7 (1–35) and PrS2-Bac7 (1–35) in E. coli BL21 (DE3). a BL21 (DE3) harboring either pColdSUMO-Bac7 (1–35) or pColdPrS2-Bac7 (1–35) was inoculated in the 5 ml of LB medium. After OD600 reached at 0.8, the 1 mM IPTG was added, and the culture was incubated overnight incubation at 16 °C. The position of PrS2-Bac7 (1–35) is indicated by an arrow. BL21 (DE3) co-transformed with pACYCmazF and pColdPrS2-Bac7 (1–35) (SPP cells) was inoculated into 5 ml of LB medium and the culture was incubated at 37 °C. Wen the OD600 reached at 0.8, the culture was transferred onto ice for 5 min, followed by 1 h incubation at 16 °C. After protein induction by the addition of 1 mM IPTG, the culture medium was incubated for overnight at 16 °C. Lane 1, SUMO-Bac7 (1–35) before induction; lane 2, SUMO-Bac7 (1–35) after induction; lane 3, PrS2-Bac7 (1–35) before induction; lane 4, PrS2-Bac7 (1–35) after induction; lane 5, SPP cells before induction; and lane 6, SPP cells after induction. b The PrS2-Bac7 (1–35) production using the SPP system and its cellular localization. The ACA-less gene for Bac7 (1–35) was expressed using pCold-PrS2 vector together with pACYCmazF at 15 °C. Lane 1, before 1 mM IPTG was added. After inducing the PrS2-Bac7 (1–35) at 15 °C for overnight, the cells was collected by centrifugation and subsequently cellular fractionation was carried out; lane 2, whole cells; lane 3, cell lysate after low speed centrifugation; lane 4, cell pellets after low speed centrifugation; lane 5, the soluble fraction, and lane 6, the insoluble fraction. c Cleavage of PrS2-Bac7 (1–35) by Factor Xa protease. The 30 μg of purified PrS2-Bac7 (1–35) in 50 μl was incubated with 4 μl of factor Xa protease at 37 °C for 4 h. After incubation, 10 μl of the reaction mixture was subjected to 20 % SDS-PAGE. Lane 1, PrS2 only; lane 2, PrS-Bac7 (1–35) before cleavage by factor Xa; lane 3, PrS2-Bac7 (1–35) after cleavage by Factor Xa; and lane M, molecular weight markers. DnaK, one of the target proteins of Bac7 (1–35), was co-purified, and shown by an arrow.
d Bac7 (1–35) purified after from PrS-Bac7 (1–35) treated by factor Xa followed by ion-exchange column chromatography. The concentration of purified Bac7 (1–35) was determined by Bradford reagent, and 1.6 μg of Bac7 (1–35) was analyzed by 20 % SDS-PAGE