Part:BBa_K3482044

azurin (UNIL) 6His

Azurin fused with a 6His tag for protein expression and purification

Usage and Biology

Azurin is a blue copper-binding redox protein originally found in the periplasmic membrane of Pseudomonas aeruginosa. It has recently been discovered to have an anticancer effect by targeting the well-known p53 apoptosis pathway and by preferentially entering cancerous cells.

We used this part to test if azurin was produced in our bacteria.

Characterization

SDS PAGE was used to show the presence or absence of azurin. For that, we used the expression strain E. coli BL21 transformed with the expression vector pET22-BBa_3482044, containing the optimized azurin coding gene mentioned above with a 6-His tag. We induced the expression using 50uM or 500uM of IPTG for 4h (Figure 1A). In a second step, we selectively purified our azurin protein using the 6-His tag in combination with HisPur Ni-NTA Magnetic Beads

Left: SDS PAGE of bacterial lysate. Samples were induced for 4h with the mentioned IPTG concentrations. The OD of all cultures was normalized to 0.5. Samples were lysed for 10min at 95°C and run on a 15% polyacrylamide gel for 30 minutes at 200V. Right: SDS-PAGE with the same culture conditions as the uninduced sample on the left. Purification performed using HisPur Ni-NTA Magnetic Beads, incubated for 5 seconds with our sample. Signaled in red is the expected azurin protein. Samples were run on a 15% polyacrylamide gel for 30 minutes at 200V.

We can observe on the left the presence of three bands around 15 kDa, which is the size range of our azurin (14kDa). Interestingly, the uninduced sample shows the highest intensity and we hypothesised that this could be due to intrinsic toxicity upon overexpression using IPTG. Thus, the leaky expression of the T7 promoter may allow for a higher yield of our protein expression. Upon purification using the specific 6-His tag, we can only detect one band (red box on the right). From these experiments, we concluded that the purification of azurin was successful.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 253
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]