Skip to main content
. 2000 Mar 21;97(8):3872–3877. doi: 10.1073/pnas.060024697

Figure 1.

Figure 1

Cloning and expression of Mb. (A) Construction of plasmid containing the Mb gene. pET13cIIFxMb was constructed by fusing the first 32 codons for the λ cII gene with the coding sequence for Mb. A cleavage site for the protease factor Xa was engineered at the junction between the cII and Mb coding sequences to allow cleavage right before the first residue of Mb. The coding sequence for the fusion protein was inserted between the NdeI and BamHI sites in vector pET13a. (B) Levels of overexpression of recombinant Mb in different expression vectors as visualized on a 5–25% PAGE gel, stained with Coomassie blue. Lane 1 shows molecular weight markers. Lane 2 contains a cell lysate of the culture containing the plasmid pUC19Mb in late saturation phase (kindly donated by S. Sligar). Lanes 3 and 4 contain lysates of cells containing the plasmid pET13Mb 3 h after induction at 37°C, in the strains BL21(DE3) and BL26(DE3), respectively. Lanes 5 and 6 contain lysates of cells containing the plasmid pET13cIIFxMb 3 h after induction at 37°C, in the strains BL21(DE3) and BL26(DE3), respectively. The level of expression is substantially higher in the T7 expression system (lanes 3–6). The plasmid pET13cIIFxMb in BL26(DE3) was used in this work.