Purification of proteins: Z-alpha and Z-omega

Piotr, Emilia

1. pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain from overnight culture inoculated in fresh LB and cultured until OD=0,5.
2. Cultures induced with different concentrations of IPTG in 22°C and 37°C.
3. Samples were collected twice: after 3h and next day (samples were centrifuged and frozen).

Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_ΔA from previous day) inoculated to liquid LB with kanamycin.

Cloning of truncated fragment of protein A (ΔA)

Michał K.

1. Optimization of PCR to obtain truncated fragment of protein A DNA.
   Primers: AL+SacI AP+NotI
   Template DNA: pDRIVE-TapTag
   Elongation time: 30s
   - Optimization of annealing temperature (gradient from 55°C to 75°C) )Fig. 1.).
   
   Fig. 1. Gradient PCR to obtain truncated protein A (temperatures: 55-75°C)
   1. Marker
   2. 50°C
   3. 55°C
   4. 60°C
   5. 65°C
   6. 70°C
   7. 75°C
   
   - Optimization of number of cycles(15, 20, 25, 30, 35) (Fig. 2.).

Fig. 2. PCR to obtain truncated protein A (various number of cycles)
1. Marker
2. 15 cycles
3. 20 cycles
4. 25 cycles
5. 30 cycles
6. 35 cycles


2. PCR to obtain truncated A protein DNA fragment.
   Primers: AL+SacI AP+NotI
   Template DNA: pDRIVE-TapTag
   Elongation time: 30s
   Annealing temperature: 60°C
   20 cycles
3. Gel electrophoresis (Fig. 3) and gel-out of 250 bp band.

Fig. 3. PCR amplified truncated protein A
1. Marker
2. PCR product (deltaA), temperature of annealing = 60°C, 20 cycles


4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI (BamHI buffer). pACYC177 vectors were also dephosphorylated.
5. Clean-up of digest reaction.
6. Gel electrophoresis for estimation of DNA concentration.
7. Overnight ligation: pACYC177+OmpA_alpha + ΔA and pACYC177+OmpA_omega + ΔA.