Dear all,
I want to express my protein in the periplasm of E. coli and decided to use the common pelB-leader-sequence for translocation to the periplasmic space.
Now I also want to experiment with the N-Terminus and attach small modifications, like
N.term-Met-Arg-Gly-Ser-[pelB]-[target protein]-C.term
I am aware that these N-terminal amino acids will be cut by the signal peptidase anyway, but they may modulate expression yield and improve half-life.
Does this modification impair the binding and effectivity of the pelB-leader sequence? How tolerant is the leader-peptide considering this? Or does even the slightest change prevent successful tranlocation to the periplasm?
Thanks for any help!
Regards,
Peter
For prediction of signal peptides, use SignalP 4.1 Server. Maybe you can test the original signal peptide and your modified one using the software to see if there is any difference, although it is just theoretically. Hope it is helpful.
It is hard to say without specifically testing, but in general you can not muck about with signal sequences very much. I also fail to see why you think it would stabilize and increase the half life since that sequence will be cleaved and not be present on the mature protein.
Yin-biao Wang: Thanks for the hint, I have designed a consensus leader-peptide using the server and will check on how it performs in experiment. :)
Michael Benedik: The very first amino-acids (that is, their codons on the mRNA-level) will critically influence translation-initiation efficiency and therefore expression yield as a whole.
Dear Peter,
you may be also interested in the following publication:
Izard et al., The Journal of Biological Chemistry 1996, 271, 21579
DOI: 10.1074/jbc.271.35.21579
http://www.jbc.org/content/271/35/21579.abstract?sid=0eac9fba-4cb4-4138-8112-01ec8e77b54c
The authors showed that the pelB leader action depends on the hydrophobicity of the core region and works best with a cationic charge in the amino-terminus.
So your modifications should not necessarily be detrimental, since these characteristics are retained in your altered leader.
To note, I found that this publication is cited in several others which aimed to improve export efficiency my modification of the leader, but I did not read those publications so far.
Best regards!
Christian
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