GFP toxicity?

G.J.A.ROUWENDAL at ato.dlo.nl G.J.A.ROUWENDAL at ato.dlo.nl
Wed Nov 29 04:54:23 EST 1995

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

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