Dear netters,
A couple of months ago I found to my surprise that GFP genes cloned in
the expression vector pTrc99A caused the host E. coli JM109 to grow
poorly in liquid culture. However, subcloning of these genes into a
modified pGEX2T vector yielded transformants that grew fine and produced
large amounts of a fluorescent fusion protein in the same host.
Recently, Haseloff and Amos (TIG 11: 328 [1995]) reported a similar
growth problem in transgenic Arabidopsis, i.e. it proved to be difficult
to regenerate fertile plants from the brightest transformants. In other
words, the same `problem' also seemed to occur in plants. This supported
my own observations on badly growing potato transgenics expressing GFP
under the control of the enhanced 35S promoter (subsequent tobacco
transformations with the same constructs were quite successful, but I
have not studied GFP expression levels yet).
Interestingly, in a recent GFP overview in TIBS 20 (448-455) by Cubitt
et al. GFP appeared to be toxic at high expression levels in two of the
listed examples of its applications. A second example of GFP toxicity in
E. coli was published by Deschamps et al. (Protein expression and
purification 6: 555-558 [1995]). These authors showed that the original
jellyfish protein appeared to be toxic, whereas a slightly altered form
was not. Combining their information with mine, I reluctantly conclude
that maybe one additional alanine at position 2 of the original GFP might
suffice to eliminate its toxicity in E. coli (and other organisms?)??
The fact that only a minority of us experienced toxicity problems may
be associated with the presence of MCS-derived aa sequences at the GFP
N-terminus in most of the constructs used until now. On the other hand,I
expect that many more of us encountered what might have been toxicity
problems (?). If so, what kind of constructs did you use (how much did
they differ from the original gene)? Do any of you have any ideas about
the mechanism behind the toxic behaviour of GFP? As far I can see, the
fluorescent character of the protein would not seem to be involved.
In summary, I'm looking forward to a lively and bright discussion.
Gerard Rouwendal (g.j.a.rouwendal at ato.dlo.nl)
Agrotechnological Research Institute ATO-DLO
P.O. Box 17
6700 AA Wageningen
The Netherlands
Tel.+31-317-475315/Fax. +31-317-412260
P.S. I apologize for any double postings as I was having some problems getting
my message posted.
