Anaerobic expression of GFP?
prigge at morel.uoregon.edu
Tue May 16 16:28:35 EST 2000
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In article <200005160819.KAA11685 at intwww.zit.med.uni-tuebingen.de>,
Wolfgang.Schechinger at med.uni-tuebingen.de ("Wolfgang Schechinger")
> Your proposal is a very good idea for studying the correct folding. I
> still want to render my GFP fluorescent, but *after* the expresseion,
> somewhat controlled. The chemistry of the chromophor is interfering
> with my experiment since it doesn't consist of amino acids.
Check out this article...
Reid, B. G. and G. C. Flynn (1997). ³Chromophore formation in green
fluorescent protein.² Biochemistry 36(22): 6786-91.
The green fluorescent protein (GFP) from the jellyfish Aequorea
Victoria forms an intrinsic chromophore through cyclization and
oxidation of an internal tripeptide motif [Prasher, D. C., et al.
(1992) Gene 111, 229-233; Cody, C. E., et al. (1993) Biochemistry 32,
1212-1218]. We monitored the formation of the chromophore in vitro
using the S65T-GFP chromophore mutant. S65T-GFP recovered from
inclusion bodies in Escherichia coli lacks the mature chromophore,
suggesting that protein destined for inclusion bodies aggregated prior
to productive folding. This material was used to follow the steps
leading to chromophore formation. The process of chromophore formation
in S65T-GFP was determined to be an ordered reaction consisting of
three distinct kinetic steps. Protein folding occurs fairly slowly
(k(f) = 2.44 x 10(-3) s(-1)) and prior to any chromophore modification.
Next, an intermediate step occurs that includes, but is not necessarily
limited to, cyclization of the tripeptide chromophore motif (k(c) = 3.8
x 10(-3) s(-1)). The final and slow step (k(ox) = 1.51 x 10(-4) s(-1))
in chromophore formation involves oxidation of the cyclized
chromophore. Since the chromophore forms de novo from purified
denatured protein and is a first-order process, we conclude that GFP
chromophore formation is an autocatalytic process.
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