leaky IPTG inducible promoters
emma at HERA.MED.UTORONTO.CA
Wed Aug 3 10:18:36 EST 1994
Does the pKK233-2 have a trc promoter and not a tac promoter like pKK233-3?
A colleague is using Invitrogen's pSE420 for expression which also has
a trc promoter. He is getting a fair amount of basal expression of his
protein but since it isn't causing the bugs any trouble he isn't too
worried about it. Expression also goes up significantly on IPTG induction
and he is consistently getting 20mg of pure protein per litre of culture.
His induction protocol is to grow the bugs into the logarithmic phase
(he prefers the OD to be closer to 1.0) then add IPTG to 1.0mm and grow
for a further 3h. What strain of bacteria are you using? If basal expression
is giving you problems you might want to consider using a strain that
carries the lacIq gene on the F' episome (JM101, 105, 109 etc., MV1184,
TG1, 2) if you aren't already.
The original reference for the trc promoter is Gene 25 (1983) 167-178
(E. Amann, J. Brosius and M. Ptashne) and they do mention some basal
expression in it (about 8% I think).
Hope this is some help
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