i did a site-directed mutagenesis of HBx gen in pET-16b plasmid to produce HBx T118N. The HBx wild-type overproduction in e.coli BL21(DE3) required IPTG 0,1 mM. On the other hand, HBx T118N could be expressed without the addition of IPTG. Have any of you experienced the same thing?
We had an issue with Luria Broth- using premixed powders for making media led to expression of proteins without IPTG. When we made LB from Tryptone, Yeast Extract and NaCl ourselves, the problem went away. This may not be an issue for you since you are using the same LB for WT and mutant. Try re-transformation and picking up another clone for the mutant. This is also another way we have done away with leaky expression.
I concur with Kartiki. Before concluding that the different expression of the two proteins is due to the mutation, it would be necessary to compare expression in the same batch of medium. Tryptone is made of a casein digest and batches are often contaminated with lactose. See papers by F. W. Studier. If leakage is a problem (e.g. because the target protein is toxic and promotes the selection of low-expressing segregants), you should use BL21(DE3)(pLysS), or better still, BL21AI (arabinose-inducible).
Dear Naufalia
I have 2 question:
1) Which Taq polimerase did you use for the site direct mutagenesis. An hi-fidelity polimerase included in the KIT?
Because in site direct mutagenesis you amplify the entire vector and if you not use a high-fidelity polimerase you can have mutations and if a mutation happen in the lac operon (present in the pet16 after the T7 promoter) or in the lacI gene, it can affect the repression.
2) The expression level of the 2 proteins, WT and mutant after induction were similar or the mutant was much more expressed?
best regards.
Manuele
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