At 07:57 AM 4/30/97 -0700, you wrote:
>SAINTIGNY Yannick 161011 wrote:
>>> Does any one known what will be the minimal deletion in the 3' end of
>>the eGFP coding sequence to knock-out the transcription and/or the
>>traduction and/or the fluorescence of the eGFP, in mammalian cell
>>transfected with the peGFP-C3 vector ( without deleting the three STOPs
>>codon). What happen if we remove the three last amino-acid of the eGFP ?
>>As far as I remember (Roger Tsien mentioned it at the last ASCB meeting),
>one can delete a maximum of about 5 AA on the c-terminus of GFP and still
>get a fluorescent protein. The situation should be the same for eGFP.
I don't recall giving a figure of 5 AA at the C-terminus of GFP in my talk,
but if I did, I would like to correct any such off-the-cuff remark and cite
the references in which the real data is published, which anyone interested
should read for themselves:
Dopf and Horiagan (1996) produced a family of genetic
truncations from the N- and C-termini. The N-terminal
methionine could be replaced by a polyhistidine tag, but
deletion of residues 2-8 prevented fluorescence or chromophore
development. The C-terminus was slightly more tolerant, in that
6 but not 13 residues could be eliminated. These narrow limits
are in good agreement with the crystal structure (Ormo et al.
1996), in which Met1 and residues 230-238 are too disordered to
Dopf, J. and Horiagon, T. (1996) Deletion mapping of Aequorea
victoria green fluorescent protein. Gene 173, 39-44.
Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y.
and Remington, S.J. (1996) Crystal structure of the Aequorea
victoria green fluorescent protein. Science 273, 1392-1395.
Roger Y. Tsien
rtsien at ucsd.edu
tel. +1(619)534-4891, fax +1(619)534-5270
Howard Hughes Med. Inst., Univ. Calif. San Diego, La Jolla, CA 92093-0647