Deletion mapping of the Aequorea victoria green fluorescent protein.
Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2-232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300-500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.
Dopf
J
J
Molecular Vaccine Laboratory, Human Gene Therapy Research Institute, Des Moines, IA 50309, USA.
Horiagon
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D037401:Scyphozoa; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
D008164:Luminescent Proteins; D009838:Oligodeoxyribonucleotides; D049452:Green Fluorescent Proteins