Figure 4
The construction of pET28a-FleA and the expression of AOL. (A) The structure of redesigned gene of FleA. (B) The detection of pET28a-FleA by enzyme digestion. Line 1 was marker DL2000; pET28a-FleA in line 2 was digested with restriction enzyme EcoR I and Xho I. (C) The BL21 cells were stimulated with 0.5 mM IPTG for 0, 1, 3, 5 and 6 h at 37 °C with shaking. The expressing quantity was the highest at the time of 5 h. The AOL corresponding to 36 KDa was detected by 8% SDS-PAGE. (D) Purification of His-tag AOL. The expressed His-tag AOL was purified by Ni-sepharose affinity chromatography. Line 1, marker; Line 2, control sample; Line 3, the purified sample with stimulation for 5 h. (E) Western blot. His-tag AOL was incubated with mAb against His-tag (1:3,000). Line 1, control sample; Line 2, the purified sample with stimulation for 5 h.
