Team:Warsaw/Calendar-Main/11 August 2008

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Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in TOP10)

Piotr, Emilia

Test was conducted in E.coli Rosetta strain expressing OmpA_omega_ΔA_alpha (with and without induction) and OmpA_A_alpha.

  1. Spinning.
  2. Suspending.
  3. Adding the lysis buffer.
  4. Boiling.
  5. Putting into poliacrylamide gel.
  6. Transfer onto nitrocellulose.
  7. Blocking.
  8. Anti-A antibody binding.
  9. Washing.
  10. Anti-rabbit antibody binding.
  11. Developing with BCIP and NBT (Fig. 1.).

Fig. 1. Detection of protein A in bacterial lysates.
1 - protein marker,
2 - E. coli without plasmid,
3, 4, 5, 6 - Omp_omega_A_alpha fusion protein:
3 - in Top10 strain with 1 mM IPTG,
4 - in Top10 strain with 0.5 mM IPTG,
5 - in Rosetta strain with 1 mM IPTG
6 - in Rosetta strain with 0.5 mM IPTG,
7 - in Rosetta strain with 0.25 mM IPTG,
8, 9, 10 - Omp_A_alpha fusion protein:
8 - in Top10 strain with 1mM IPTG,
9 - in Rosetta strain with 1mM IPTG,
10 - in Rosetta strain with 0.5 mM IPTG,

We didn't observe differences in expression and degradation in Rosettas nor in TOP10. Therefore we suppose that degradation of the fusions is caused by factor other than Lon and OmpT proteases.

Purification of proteins: Z-alpha and Z-omega

Emilia

100 ml of medium inoculated with overnight culture, then used as inoculum for 1 l culture in presence of ampicillin, kanamycin and IPTG (according to protocol).

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