Currently I am working on beta-lactamase enzyme. Proteins are well expressed and purified (plasmid pET28a)but not getting result of MIC in BL21/Top10 E.coli host cells while treating with Beta-lactam antibiotics. Protocol I am following is according to CLSI Guideline. Out of 8, one or two same vector constructs containing other beta-lactamase gene is performing well in MIC experiments.
What could be the possible reasons?
Are you expressing them with their endogenous signal sequence and without any N-terminal additions? You can not add an N-terminal tag onto a signal sequence.
When you say you have expression, do you know the protein is in the periplasm? beta lactamase needs to be periplasmic in order to function.
Thank you so much Prof. Michael for your informative answer. We have deleted the N-terminal signal sequence of the proteins and cloned them in pET-28a vector for further downstream experiments and used C-terminal His-tag to purify the proteins through Ni-NTA affinity chromatography. We have not used any N-terminal tag itself.
As we have deleted the signal sequence, hopefully the protein would be in cytoplasm not in periplasm.
Please help me out what the next step should be to get MIC result.
Beta lactamase does not work in the chtoplasm, it needs to be secreted to the periplasm in order to be active. So it will not be active without a signal sewience.
Thank you so much Prof. Michael for your answer.
It will be very helpful for my further proceedings.
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