Revision as of 16:48, 19 September 2012

Hepatitits B core antigen with IPTG induced promotor

This construct produces Hepatitis B core antigen subunits. These subunits can be used to make HepBcAg Virus-Like Particles. The promotor and RBS that have been added to the gene allow induced expression of the subunits which increases the yield.

This BioBrick can serve as a template for modifications in the external loop of the VLP.

Protolcols

Transform an E. coli strain that is equipped for protein expression (e.g. BL21) with the BioBrick.

Growing Culture

Reagents & Materials

For a 50 mL culture:

reagents:

• 60 mL of LB
• antibiotics (C)
• 0.25 mL of IPTG stock solution 250 mM
• MQ-water

materials:

• Pipettes and pipettes points
• 250 ml Erlenmeyer
• Greiner tubes
• Freezer
• Centrifuge for Greiner tubes

Procedure

1. Prepare a Erlenmeyer flask with 50mL of LB-medium and keep it at 37°C
2. Pick E.coli BL21 from a plate and grow them over night in a 10 mL LB culture containing 50mg/L antibiotics (chloramphenicol) at 37°C
3. Inoculate the large flask with 1 mL of the overnight culture and grow at 37°C for 2.5h until the OD600 reaches approx. 0.6
4. Induce with 1.25 mM IPTG (0.25 mL of a 250 mM stock)
5. Incubate at 37°C for 4 h
6. Centrifuge the cells in 50ml greiner tubes at 4700 rpm for 18 minutes
7. Decant the supernatant and resuspend the pellets in ± 10 mL washing buffer each
8. Centrifuge again at 4700 rpm for 18 minutes
9. Decant the supernatant and centrifuge for another minute
10. Pipette any liquid to clear the tube of supernatant
11. (possible to freeze the cells to -20°C at this point)

Dialysis of the VLPs

Reagents & Materials

Reagents:

• Formation Buffer
• Formation Buffer + 8M urea

Materials:

• Pipettes + pipettepoints
• Greinertubes
• Dialysis tubing
• Vortex
• French Press
• Centrifuge for Greiner tubes
• Sorval or a similar centrifuge
• Coldroom

Procedure

The temperature of the VLP’s is very important. All the work should be done in the cold room (4°C) and the used tubes have to be on ice.

1. Resuspend the cells in 5 mL of Disassembly buffer
2. Disrupt the cells by french press, cell pressure: 1000
3. Add Disassembly buffer to 25 mL total volume
4. Centrifuge the lysate until the insoluble fraction is pelleted – 4°C, 4700 rpm, 18 min
5. Remove the supernatant completely and dissolve the pellet in 10 mL of cold Disassembly buffer containing 8 M urea (about 10-15 min). Vortex the pellet to easily resuspend the while cooling on ice. Do not allow the buffer to heat up above 25°C
6. Allow the pellet to dissolve for 2-5 minutes
7. Dilute the 10 mL of 8 M urea/ Disassembly buffer with 15 mL Disassembly buffer. If crystals form, the batch should be discarded
8. Pellet everything that is not dissolved by centrifuging at 15000 rpm at 4°C for 15 minutes (Sorvall ultracentrifuge in the basement)
9. Transfer the supernatant to a clean 50 mL Greiner tube
10. Prepare a piece of dialysis tubing with a large diameter by soaking it in cooking demiwater until it opens up
11. Put a tight knot on one side and fill the tubing with the supernatant
12. Knot the tube on the other side leaving a small air bubble inside
13. Dialyse the tube (about 25 mL volume) against 1x Dialysis buffer for 4 hours or overnight
14. Dialysis is performed 6 times against 1x Formation(4h per dialysis, or overnight)

Purifying the VLPs

Materials:

• Pipettes + Pipette tips
• Eppendorf tubes
• Greiner tubes
• Centrikon or a similar ultracentrifuge + all additional equipment

Procedure

1. Cut the knot of the dialysis tubing and empty the content into a clean 50 mL Greiner tube
2. Prepare a Centrikon T-1055 ultracentrifugation vessel and use a TFT 65.13 rotor, fill the vails with 4 ml of the sample
3. Gently put 4 mL of a 20% Sucrose in demiwater solution under the 4 mL sample
4. Balance the vails with reasseblybuffer
5. Centrifuge at 45000 rpm for 3 h, remember the orientation of the vessel so you know where to look for the pellet
6. Take a sample from the supernatant (for Western blotting) and decant the rest (supernatant also contains single subunits)
7. A transparent pellet should be visible
8. Resuspend/ dissolve the pellet in 200 uL of Formation Buffer

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Sequence and Features