A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.
We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.
Gurskaya
Nadya G
NG
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
Fradkov
Arkady F
AF
Pounkova
Natalia I
NI
Staroverov
Dmitry B
DB
Bulina
Maria E
ME
Yanushevich
Yurii G
YG
Labas
Yulii A
YA
Lukyanov
Sergey
S
Lukyanov
Konstantin A
KA
D000818:Animals; D019556:COS Cells / Q000166:cytology; D002460:Cell Line; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011993:Recombinant Fusion Proteins; D037401:Scyphozoa / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014162:Transfection
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins