A novel yellowish-green fluorescent protein from the marine copepod, Chiridius poppei, and its use as a reporter protein in HeLa cells.
A crustacean gene, encoding for a new class of GFP-like protein, has been isolated from a cDNA library of the deep-sea (benthic) copepod crustacean, Chiridius poppei, by expression cloning. The cDNA library was constructed in a pBluescript II vector and screened using a non-UV transilluminator, obtaining a positive clone. The clone consisted of a 781-bp fragment of cDNA with a 660-bp open reading frame, which encoded for a 219-amino acid polypeptide with a calculated molecular mass of 24.7 kDa. The protein was overexpressed in Escherichia coli, purified to homogeneity by anion-exchange and size-exclusion chromatographies. The protein, CpYGFP, had excitation and emission maxima at 507 and 517 nm, respectively. CpYGFP existed as a dimer in solution and could be expressed either alone or as a fusion protein in HeLa cells. Dual labeling experiments carried out with CpYGFP-actin and DsRed2-Nuc demonstrated the usefulness of CpYGFP as a reporter in the subcellular localization of actin.
VALWAY Technology Center, NEC Soft, Ltd., 1-18-7, Tokyo 136-8627, Japan. hiromi-masuda@aist.go.jp
Hiromi
H
Masuda
Yasuhiro
Y
Takenaka
Atsushi
A
Yamaguchi
Satoshi
S
Nishikawa
Hiroshi
H
Mizuno
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D033342:Copepoda / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000302:isolation & purification; D015870:Gene Expression; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA
D000199:Actins; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins