kbsb7s04 at hkem.com ("Rainbow") wrote:
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::We have recently cloned a cryptic gene from Burkholderia. It can =
:constitutively be expressed by its own promoter in pSP73 backbone.
::However, when I subcloned this particular gene into expression vectors =
:using tac, lac, T7 or T5 promoter, the expression level was the same or =
:even less than those expressed in pSP73 backbone.
::I have tried to optimize the expression conditions like different temp, =
:sub-culture volume, IPTG concentration......using some strains that are =
:able to deal with rare codon usage problem.
Sounds like a situation where a gene is toxic and is selected
against during growth/expression. Two main remedies then:
tight, _very_ tight repression to ensure promotor does not leak
in the absence of induction and never ever an overnight culture
but rather plate to culture directly.
:Usually, the amount of protein being expressed are too minute to be =
:observed as a sharp band in SDS-PAGE. I could only managed to get 2 =
:trials that roughly 10% of my protein out of total protein were =
:expressed.
On the other hand 10% of total protein is a lot, and many people
in many cases are quite happy enough when they can get 1% of
of total protein.
- Dima
