Article Figures & Data
Figures
- Fig 1
Schematic presentation of the MazE-MazF fusion protein with a linker containing a specific cleavage site for factor Xa, HIV PR, or HCV NS3 protease. (A) Schematic presentation of the MazE-MazF fusion protein with a linker (in green) containing a protease cleavage site and a His6 tag at the C-terminal end. (B) The amino acid sequence of MazE. The secondary structures, determined by X-ray crystallography (11), are also indicated above the sequence. Note that it contains a 22-residue-long unstructured C-terminal extension. β-Strands and α-helices are indicated with blue arrows and rectangular shapes, respectively. Red arrows indicate the starting residues for the C-terminal segments used in the present study. The residue number of the first residue of each segment is also indicated. (C) Crystal structure of the MazE-MazF complex (Protein Data Bank entry 1UB4). Black arrows and numbers indicate the sites and the residue numbers of truncated MazE fragments. (D) Schematic presentation of the activation of the MazE-MazF fusion protein having a protease cleavage site in the linker. Blue and red molecules of the MazE-MazF fusion protein form a dimer as shown, and the yellow box indicates the protease cleavage site between the MazE fragment and MazF.
- Fig 2
Characterization of MazE-MazF fusion protein. (A) Toxicity of MazE(42)F-FXa in E. coli. The transformants harboring pBAD24 vector (1 and 8), mazF-ec (2 and 7), mazF-bs (4 and 5), and mazE(42)F-FXa (3 and 6) were streaked on M9 plates with or without 0.2% arabinose and incubated at three different temperatures, 24, 37, and 42°C. (B) Gel filtration of MazE(42)F-FXa. The linear trend line is used for calculating the molecular mass for the MazE-MazF fusion protein. The purification of MazE(42)F-FXa (C), MazEF-HIV (D), and MazEF-HCV (E) are also shown. Lane 1, molecular mass markers; lane 2, the whole-cell lysate without induction; lane 3, the whole-cell lysate after incubation for 4 h at 37°C; lane 4, the cell pellet; lane 5, flowthrough fraction; lane 6, wash fraction; and lanes 7 to 15, elution fractions. Arrows indicate the positions of monomers of the fusion proteins, and arrowheads indicate the positions of their dimers.
- Fig 3
Activation of MazF from MazE(42)F-FXa, MazEF-HIV, and MazEF-HCV proteins. (A) Cleavage of MazEF-HIV. Black arrows indicate, from top to bottom, MazE-MazF fusion protein, MazF, and HIV PR. Lane 1, molecular mass markers; lane 2, HIV PR; lane 3, MazE-MazF fusion protein; lane 4, HIV PR by itself incubated for 30 min; lane 5, MazE-MazF fusion protein by itself incubated for 30 min. For lanes 6 to 15, the concentration of HIV PR was increased five times for each lane with a constant amount of the MazE-MazF fusion protein (280 μM). Thus, the molar ratios between HIV PR and MazE-MazF fusion protein changed from 3 × 10−6:1 (lane 6) to 1.6:1 (lane 15). The activation of MazF from the MazE-MazF fusion protein by factor Xa, HIV PR, and HCV NS3 protease is shown in panels B, C, and D, respectively. The MazF activity was measured with MS2 phage RNA as described previously (17).
Tables
- Table 1
Activation of various MazE-MazF fusion proteins by factor Xa, HIV-1 PR, and HCV NS3 protease
Name MazE regiona (aa) Protease cleavage site Expressionb Toxicityc MazEF-FXa 42 to 82 Factor Xa +++ − MazE(61)F-FXa 61 to 82 Factor Xa + + MazE(65)F-FXa 65 to 82 Factor Xa − +++ MazE(71)F-FXa 71 to 82 Factor Xa − +++ MazEF-HIV 42 to 82 HIV-1 PR +++ − MazEF-HCV 42 to 82 HCV NS3 protease +++ − ↵a The residue numbers from the N-terminal end of MazE are shown (11).
↵b A plus sign indicates expression, and three plus signs indicate very high expression of the fusion proteins. A minus indicates no expression.
↵c A minus indicates that the fusion protein is not toxic even in the presence of IPTG. A plus sign indicates that it is toxic only in the presence of IPTG. Three plus signs indicate that it is toxic even in the absence of IPTG.
