Hi, all.
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.
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.
Did anyone encounter the same problem and can give me some suggestion? Many Thanks!
Kenneth
Graduate student
Email: kbsb7s04 at hkem.com
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